Team:Heidelberg/notebook/cf
week number 42
▼2015-10-18 In vitro transcription of P1, P2
For every reaction:
|
Stock solution |
Final concentration |
[µl] |
|
ATP 100mM |
4 mM |
8 |
|
CTP 100mM |
4 mM |
8 |
|
UTP 100mM |
4 mM |
8 |
|
GTP 100mM |
4 mM |
8 |
|
DTT 1M |
1 mM |
2 |
|
DMSO |
5% |
10 |
|
10x Transcription buffer |
1x |
20 |
|
DNA |
1 µg |
20 |
|
ddH20 |
|
106 |
|
T7 RNA polymerase |
|
3 |
- Reaction was incubated for 3 h at 37 °C
- After 1.5 h another 2 µL T7 RNA Polymerase were added
- Addition of 2 µL DNase I and further incubation at 37 °C for 20 min
- The constructs containing hammerhead- or HDV-ribozymes was heated up to 95° for 5 min, so that there is a coplete cleavage.
RNA purification by precipitation:
- Samples was mixed with 200 µL of 2 x loading dye and purified over a 10 % PAGE
- Bands were visualized by UV shadowing and suitable bands were excised
- RNA was eluted out of the gel using 0.3 M NaAc pH 5.5 in three elution steps
- Gel parts were filtered of and 2.5 volumes of -20 °C EtOH were added, sample was stored at -20 °C oN to let the RNA precipitate
- Spin sample at 16,000 g for 30 min, discard the supernatant
- Washed the pellet twice with 70 % EtOH and dissolved RNA in 20 µL of MQ water
week number 37
▼2015-09-07 Cloning of ribozyme 12-1 (Ribozyme without Insert) from pSB1C3 into p413-GPD
Procedures:
Digestion:
Description:
Materials and Chemicals:
4 µl of pSB1C3 with pcat-MCS and ribozyme 12, about 2 µg of DNA in total
2 µl Cutsmart Buffer
1 µl BamI-HF
1 µl SalI-HF
12 µl ddH2O
The digest was incubated for 1 hour at 37°C while mixing slowly
The lower band with cut out insert (ribozyme 12) was purificated by gel extraction
Ligation:
Description:
Materials and Chemicals:
1 µl of p413-Vector DNA
6 µl of ribozyme 12 insert (purificated by gel extraction)
1 µl of T4-Ligase
2 µl of T4-Ligase Buffer
10 µl ddH2O
The ligation was incubated for 30 minutes at room temperature (about 25°C)
KCM Transformation:
Description:
Steps:
- Take 50µl chemical competent E. coli from -80 freezer and thaw on ice
- Add (as master mix):
2,5µl DNA
10µl KCM 5x
37,5µl H2O
- Incubate on ice for 30 minutes
- Heat shock at 42°C for 1 minute
- Incubate on ice for 2 minutes
- Add 900 µl of LB or 2x YT Medium
- Incubate on 37°C for 60min
- Centrifuge 5min at 1000g
- Take 900µl of supernatant and throw away
- Resuspend pellet in remaining media
- Plate out on agar with antibiotics (1:1 / 1:10)
Used DNA: Ligation of p413 and ribozyme 12-1
10 µl instead of 2,5 µl were used
▼2015-09-09 PCR of Type-1 Ribozymes for in vitro assay
|
|
Ribozyme CFTR1 A |
[µl] |
Ribozyme CFTR1 C |
[µl] |
Ribozyme CFTR1 T |
[µl] |
|
Primer Fwd |
DH_82 |
5 |
DH_82 |
5 |
DH_82 |
5 |
|
Primer Rev |
DH_33 |
5 |
DH_83 |
5 |
DH_83 |
5 |
|
Template |
Ribozyme CFTR1 DE A |
0,5 |
Ribozyme CFTR1 DE C |
0,5 |
Ribozyme CFTR1 DE T |
0,5 |
|
ddH2O |
|
14,5 |
|
14,5 |
|
14,5 |
|
Q5 Polymerase |
|
25 |
|
25 |
|
25 |
Conditions:
|
Step |
Temperature [°C] |
Time |
Cycles |
|
Initial denaturation |
98 |
1:00 |
1 |
|
Denturation |
98 |
0:10 |
35 |
|
Annealing |
66 |
0:15 |
|
|
Extension |
72 |
0:15 |
|
|
Final Extension |
72 |
1:00 |
1 |
|
Hold |
4 |
|
Cycles |
After PCR DNA was purified by Qiagen PCR Purification Kit ans stored at -20°C.
▼2015-09-10 In vitro transcription of CFTR1-type ribozymes
For every reaction:
|
Stock solution |
Final concentration |
[µl] |
|
ATP 100mM |
4 mM |
8 |
|
CTP 100mM |
4 mM |
8 |
|
UTP 100mM |
4 mM |
8 |
|
GTP 100mM |
4 mM |
8 |
|
DTT 1M |
1 mM |
2 |
|
DMSO |
5% |
10 |
|
10x Transcription buffer |
1x |
20 |
|
DNA |
1 µg |
20 |
|
ddH20 |
|
106 |
|
T7 RNA polymerase |
|
3 |
- Reaction was incubated for 3 h at 37 °C
- After 1.5 h another 2 µL T7 RNA Polymerase were added
- Addition of 2 µL DNase I and further incubation at 37 °C for 20 min
- Reaction was heated up to 95° for 5 min, so that there is a coplete cleavage.
RNA purification by precipitation:
- Samples was mixed with 200 µL of 2 x loading dye and purified over a 10 % PAGE
- Bands were visualized by UV shadowing and suitable bands were excised
- RNA was eluted out of the gel using 0.3 M NaAc pH 5.5 in three elution steps
- Gel parts were filtered of and 2.5 volumes of -20 °C EtOH were added, sample was stored at -20 °C oN to let the RNA precipitate
- Spin sample at 16,000 g for 30 min, discard the supernatant
- Washed the pellet twice with 70 % EtOH and dissolved RNA in 20 µL of MQ water
week number 35
▼2015-08-25 Plasmid prep of colonies containing c(p413-GPD; sfGFP_delY)
Goal: Verifying the sequences of the picked cultures
- Plasmids of the cultures grown overnight were purified according to the manufacturers protocol.
- Sequences were confirmed as correct by sequencing for colonies 3-2, 4-1,4-2 and 5-1.
- Culture 4-1 was used for further experiments.
week number 34
▼2015-08-17 PCR of Part 1 and 2 of the CFTR2 Ribozyme Insert for in vitro transcription
|
|
P1 |
[µl] |
P2 |
[µl] |
|
Primer Fwd |
DH_54 |
5 |
DH_56 |
5 |
|
Primer Rev |
DH_55 |
5 |
DH_57 |
5 |
|
Template |
Insert CFTR2 |
0,5 |
Insert CFTR2 |
0,5 |
|
ddH2O |
|
14,5 |
|
14,5 |
|
Q5 Polymerase |
|
25 |
|
25 |
Conditions:
|
Step |
Temperature [°C] |
Time |
Cycles |
|
Initial denaturation |
98 |
1:00 |
1 |
|
Denturation |
98 |
0:10 |
35 |
|
Annealing |
66 |
0:15 |
|
|
Extension |
72 |
0:15 |
|
|
Final Extension |
72 |
1:00 |
1 |
|
Hold |
4 |
|
|
After PCR DNA was purified by ethanol precipitation and stored at -20°C
▼2015-08-19 Labeling of P1 with G-Azide
Reaction was performed with 2-fold concentration of labeling nucleotide and a longer incubation time in comparison to first labeling experiment.
|
|
Reaction [µl] |
Control without PAP[µl] |
Control without template [µl] |
|
PAP (Poly-A-Polymerase, yeast) |
1 |
/ |
1 |
|
RNAse inhibitor |
1 |
1 |
1 |
|
PAP buffer 5x |
4 |
4 |
4 |
|
Nucleotide G-Azide, 100µM |
4 |
4 |
4 |
|
P1-RNA |
5 (= 50µM) |
5 (= 50µM) |
/ |
|
ddH2O |
5 |
6 |
10 |
- Reaction was incubated for 2 h at 37 °C
- Heat inactivation at 65°C for 10min
- Purification by ethanol precipitation
▼2015-08-20 Splinted Ligation of modified P1 and P2
|
|
Reaction [µl] |
Control without Ligase [µl] |
Control without Splint [µl] |
|
P1 RNA, labeled |
6 (=1µM) |
6 (=1µM) |
6 (=1µM) |
|
P2 RNA |
0,5 (=0,5µM) |
0,5 (=0,5µM) |
0,5 (=0,5µM) |
|
Splint |
0,7 (=2,25µM) |
0,7 (=2,25µM) |
/ |
|
10x Ligation buffer |
3 |
3 |
3 |
|
T4 DNA ligase |
1 |
/ |
1 |
|
ddH2O |
13,8 |
15,8 |
15,5 |
- Reaction was heaten up to 95°C for 30s without T4 DNA ligase and then cooled down to room temperature
- After 15min T4 DNA ligase was added.
- Reaction was incubated for 1 h at 37 °C
- Heat inactivation at 80°C for 10min
▼2015-08-20 Copper Click – Test of Labeled Part 1 and Splinted ligation
To check if the azide-modified NTPs are incorporated the reactive azide is clicked to an alkyne activated FAM-alkyne fluorophore under copper catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC). Samples are after the reaction separated on a 20 % denaturing PAGE and visualized first in the fluorescent channel for FAM and afterwards stained with SYBR
Gold to visualize the RNA. Controls were performed as well.
Reaction conditions:
|
cStock |
cFinal |
V[µL] |
|
|
Phosphate buffer pH 7.0 |
100 mM |
50 mM |
12,5 |
|
FAM alkyne |
10 µM |
400 nM |
1 |
|
Azide modified RNA |
1 µM |
200 nM |
5 |
|
Cu(II)SO4 |
2 mM |
100 µM |
1.25 |
|
THPTA |
5 mM |
500 µM |
2.5 |
|
Sodium ascorbate |
10 mM |
1 mM |
2.5 |
|
H2O |
Ad 25 |
0.25 |
|
|
Final |
|
25 |
Conditions as Winz, 2012.
- All compounds were mixed in the given order
- Cu(II)SO4 and THPTA and H2O were mixed before adding to the mixture to let THPTA chelat the Cu
- Lastly sodium ascorbate was added to reduce the Cu(II) to Cu(I)
- Reaction was incubated for 1 h at 37 °C and afterwards put at -20 °C
Checking success on a 20 % denaturing PAGE
- Samples were mixed with an appropriate amount of 2 x loading dye and separated on a 20 % denaturing PAGE
- Gel was scanned with a Biorad ChemDoc, GE in fluorescent mode using the pre-set FAM parameters
- Afterwards gel was stained using SYBR gold and scanned with the Typoon in SYBR Gold mode
Result and Outlook:
Labeling of RNA with azide and splinted ligation was also at the second tryonly partially successful. The repair-oligonucleotide is ordered as RNA-synthesis.
▼2015-08-22 Mutation PCR of pMaM17
Goal: Deleting a Y in sfGFP from pMaM17 rendering it non-functional.
- PCR with primers xxhb003xx and xxhb004xx was performed using a standard protocol with 68°C as annealing temperature.
- PCR was performed using Phusion Flash MasterMix.
- 300 µl were split amongst 4 tubes.
- PCR was followed by a DpnI digest.
- Samples were purified by gel extraction according to manufacturs protocol.
- NanoDrop showed a concentration of 743 ng/µl after purification, with 29 µl sample left to use.
▼2015-08-22 Digestion of pMaM(sfGFP_delY)
Goal: Digest pMaM17(sfGFP_delY) with BamI and SalI
- 5 µl of previous PCR product (pMaM17(sfGFP_delY) were added to 11 µl ddH20, 1 µl BamI, 1 µl SalI and 2 µl CutSmart buffer.
- Sample rested for 1 hour at room temperature.
- Heat inactivation was performed at 95 °C for 1 min.
- Purification was performed according to manufacturers protocol.
▼2015-08-22 Digestion of p413-GPD
Goal: Digest p413-GPD with BamI and SalI
- 3 µl of p413-GPD plasmid were added to 13 µl ddH20, 1 µl BamI, 1 µl SalI and 2 µl CutSmart buffer.
- Sample rested for 1 hour at room temperature.
- Heat inactivation was performed at 95 °C for 1 min.
- Linearized plasmid was purified by performing gel extraction according to manufacturers protocol.
▼2015-08-22 Ligation of p413-GPD_digested and sfGFP_delY_digested
Goal: Ligation of p413-GPD and sfGFP_delY, both previously digested with BamI and SalI
- 100 ng of digested p413-GPD plasmid and 500 ng of digested sfGFP_delY were ligated using T4 DNA ligase in a 20 µl assay.
- Sample rested for 1 hour at room temperature.
- Purification was performed according to manufacturers protocol.
- NanoDrop determined a concentration of 18.9 ng/µl with 19 µl sample remaining.
▼2015-08-22 Transformation of E. coli with sfGFP_delY in p413-GPD
Goal: Transforming E. coli with sfGFP_delY inserted into p413-GPD after a digest with BamI and SalI
-
4 x 50 µl of chemically competent E. coli were thawed on ice
-
4 samples were prepared, each containing 10 µl KCM.
-
Ligated plasmid was added as 5 µl, 4 µl, 3 µl and 2 µl, one volume per sample.
-
Samples were filled up to 50 µl with ddH20.
-
50 µl of hawed cells were added to each sample.
-
Samples were held at 42 °C for 2 min.
- Samples were incubated on ice for 2 min
- 900 ul of media was added to each sample.
- Samples were incubated at 37 °C for 60 min.
- Cells were sedimented by centrifuging at 1000 g for 5 min.
- Supernatant was removed.
- Cells were resuspended in media.
- Cells were spread on one plate each.
- Cells incubated overnight at 37°C.
▼2015-08-23 Colony picking of E. coli transfected with c(p413-GPD; sfGFP_delY)
Goal: Confirming a culture containing the correct plasmid with sfGFP_delY ligated into p413-GPD.
- Colonies were picked from plates
-
Concetration of plasmid in spread sample Picked colonies 2 µl 1 3 µl 2 4 µl 3 5 µl 2 -
Picked colonies were grown overnight as a liquid culture.
week number 33
▼2015-08-10 PCR of Part 1 and 2 of the CFTR2 Ribozyme Insert for in vitro transcription
Durchführung
|
|
P1 |
[µl] |
P2 |
[µl] |
|
Primer Fwd |
DH_54 |
5 |
DH_56 |
5 |
|
Primer Rev |
DH_55 |
5 |
DH_57 |
5 |
|
Template |
Insert CFTR2 |
0,5 |
Insert CFTR2 |
0,5 |
|
ddH2O |
|
14,5 |
|
14,5 |
|
Q5 Polymerase |
|
25 |
|
25 |
Conditions:
|
Step |
Temperature [°C] |
Time |
Cycles |
|
Initial denaturation |
98 |
1:00 |
1 |
|
Denturation |
98 |
0:10 |
35 |
|
Annealing |
66 |
0:15 |
|
|
Extension |
72 |
0:15 |
|
|
Final Extension |
72 |
1:00 |
1 |
|
Hold |
4 |
|
|
After PCR DNA was purified by ethanol precipitation and stored at -20°C
▼2015-08-10 PCR of Ribozyme target for in vitro assay
|
|
Target |
[µl] |
|
Primer Fwd |
DH_58 |
5 |
|
Primer Rev |
DH_59 |
5 |
|
Template |
CFTRtestconstruct |
0,5 |
|
ddH2O |
|
14,5 |
|
Q5 Polymerase |
|
25 |
Conditions:
|
Step |
Temperature [°C] |
Time |
Cycles |
|
Initial denaturation |
98 |
2:00 |
1 |
|
Denturation |
98 |
0:20 |
35 |
|
Annealing |
66 |
0:20 |
|
|
Extension |
72 |
1:00 |
|
|
Final Extension |
72 |
3:00 |
1 |
|
Hold |
4 |
|
Cycles |
After PCR DNA was purified by Qiagen PCR Purification Kit ans stored at -20°C.
▼2015-08-11 In vitro transcription of P1, P2, Ribozyme 2 A/C/T and Ribozyme Target
For every reaction:
|
Stock solution |
Final concentration |
[µl] |
|
ATP 100mM |
4 mM |
8 |
|
CTP 100mM |
4 mM |
8 |
|
UTP 100mM |
4 mM |
8 |
|
GTP 100mM |
4 mM |
8 |
|
DTT 1M |
1 mM |
2 |
|
DMSO |
5% |
10 |
|
10x Transcription buffer |
1x |
20 |
|
DNA |
1 µg |
20 |
|
ddH20 |
|
106 |
|
T7 RNA polymerase |
|
3 |
- Reaction was incubated for 3 h at 37 °C
- After 1.5 h another 2 µL T7 RNA Polymerase were added
- Addition of 2 µL DNase I and further incubation at 37 °C for 20 min
- The constructs containing hammerhead- or HDV-ribozymes was heaten up to 95° for 5 min, so that there is a coplete cleavage.
RNA purification by precipitation:
- Samples was mixed with 200 µL of 2 x loading dye and purified over a 10 % PAGE
- Bands were visualized by UV shadowing and suitable bands were excised
- RNA was eluted out of the gel using 0.3 M NaAc pH 5.5 in three elution steps
- Gel parts were filtered of and 2.5 volumes of -20 °C EtOH were added, sample was stored at -20 °C oN to let the RNA precipitate
- Spin sample at 16,000 g for 30 min, discard the supernatant
- Washed the pellet twice with 70 % EtOH and dissolved RNA in 20 µL of MQ water
▼2015-08-11 Labeling of P1 with G-Azide
|
|
Reaction [µl] |
Control without PAP[µl] |
Control without template [µl] |
|
PAP (Poly-A-Polymerase, yeast) |
1 |
/ |
1 |
|
RNAse inhibitor |
1 |
1 |
1 |
|
PAP buffer 5x |
4 |
4 |
4 |
|
Nucleotide G-Azide, 100µM |
2 |
2 |
2 |
|
P1-RNA |
3 (= 5µM) |
3 |
/ |
|
ddH2O |
9 |
10 |
12 |
- Reaction was incubated for 2 h at 37 °C
- Heat inactivation at 65°C for 10min
- Purification by ethanol precipitation
▼2015-08-12 Splinted Ligation of modified P1 and P2
|
|
Reaction [µl] |
Control without Ligase [µl] |
Control without Splint [µl] |
|
P1 RNA, labeled |
12 (=0,5µM) |
12 (=0,5µM) |
12 (=0,5µM) |
|
P2 RNA |
0,5 (=0,5µM) |
0,5 (=0,5µM) |
0,5 (=0,5µM) |
|
Splint |
0,7 (=2,25µM) |
0,7 (=2,25µM) |
/ |
|
10x Ligation buffer |
3 |
3 |
3 |
|
T4 DNA ligase |
1 |
/ |
1 |
|
ddH2O |
11,8 |
12,8 |
12,5 |
- Reaction was heaten up to 95°C for 30s without T4 DNA ligase and then cooled down to room temperature
- After 15min T4 DNA ligase was added.
- Reaction was incubated for 1 h at 37 °C
- Heat inactivation at 80°C for 10min
▼2015-08-13 Transfer of the ribozymes and inserts into yeast and mammalian vectors
Procedures:
Digestion of pSB1C3 + MCS +pcat with and without ribozymes:
Description
Reaction mix:
1 µg of DNA
0,1 µl of BamHI-HF
0,1 µl of SalI-HF
2 µl Cutsmart
ad 20 µl ddH2O
Digestion for 1 hour at 37°C
After the reaction the samples were given on a 0,8% Agarose gel and were then purified by gel extraction.
Ligation of the ribozymal fragments into p413
Description:
Chemicals:
1 µl of Vector DNA (about 25 ng) dephosporylated
5 µl of Insert DNA (about 10 ng)
2 µl T4-Ligation Buffer
1 µl T4-Ligase
10 µl ddH2O
The Mix was incubated for 30 minutes at room temperature
Transformation of p413 + Ribozymes + Inserts:
Description:
Steps:
- Take 50µl chemical competent E. coli from -80 freezer and thaw on ice
- Add (as master mix):
2,5µl DNA
10µl KCM 5x
37,5µl H2O
- Incubate on ice for 30 minutes
- Heat shock at 42°C for 1 minute
- Incubate on ice for 2 minutes
- Add 900 µl of LB or 2x YT Medium
- Incubate on 37°C for 60min
- Centrifuge 5min at 1000g
- Take 900µl of supernatant and throw away
- Resuspend pellet in remaining media
- Plate out on agar with antibiotics (1:1 / 1:10)
The prior ligations were used for the transformation.
Colony PCR of ribozymes + Inserts:
|
96-well |
||||||||||||
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
|
|
1 |
1-2a |
1-2d |
3-4a 1 |
3-4a 2 |
3-4a 3 |
3-4b 1 |
3-4b 2 |
3-5b 1 |
3-5b 2 |
3-5b 3 |
||
|
2 |
4-1a 1 |
4-1a 2 |
7-2b |
4-1b 1 |
4-1b 2 |
4-1b 3 |
5-2c |
6-1b 1 |
6-1b 2 |
6-1b 3 |
6-1c |
6-1d |
|
3 |
7-2a 1 |
7-2a 2 |
4-1a 3 |
7-2e |
8-1a 1 |
8-1a 2 |
8-1a 3 |
9-1a |
9-1b 1 |
9-1b 2 |
9-1b 3 |
|
|
4 |
10-3a 1 |
10-3a 2 |
10-3a 3 |
10-3c 1 |
10-3c 2 |
10-3c 3 |
10-5a 1 |
10-5a 2 |
10-5a 3 |
11-1c 1 |
11-1c 2 |
11-1c 3 |
|
5 |
12-1b 1 |
12-1b 2 |
12-1b 3 |
12-1c 1 |
12-1c 2 |
12-1c 3 |
12-1d 1 |
12-1d 2 |
12-1d 3 |
13-1a 1 |
13-1a 2 |
13-1a 3 |
|
6 |
13-1b 1 |
13-1b 2 |
13-1b 3 |
14-1c 1 |
14-1c 2 |
14-1c 3 |
14-1e 1 |
14-1e 2 |
14-1e 3 |
15-2b 1 |
15-2b 2 |
15-2b 3 |
|
7 |
15-3a 1 |
15-3a 2 |
15-3a 3 |
15-3b 1 |
15-3b 2 |
15-3b 3 |
15-3d 1 |
15-3d 2 |
15-3d 3 |
16-3c 1 |
16-3c 2 |
16-3c 3 |
|
8 |
16-3e |
16-3d 1 |
16-3d 2 |
16-3d 3 |
3 Colonies were picked from every plate which showed 3 or more colonies.
Colonies in the plates after transformation:
|
Ribozyme/Colony |
Number of colonies |
|
1-2a |
1 |
|
1-2d |
1 |
|
1-2e |
0 |
|
3-4a |
>3 |
|
3-4b |
2 |
|
3-4d |
0 |
|
3-5a |
0 |
|
3-5b |
>3 |
|
3-5e |
0 |
|
4-1a |
>3 |
|
4-1b |
3 |
|
5-2c |
1 |
|
5-2e |
0 |
|
6-1b |
>3 |
|
6-1c |
1 |
|
6-1d |
1 |
|
7-2a |
2 |
|
7-2b |
1 |
|
7-2c |
0 |
|
7-2e |
1 |
|
8-1a |
3 |
|
9-1a |
1 |
|
9-1b |
3 |
|
9-1c |
0 |
|
10-2a |
0 |
|
10-3a |
3 |
|
10-3c |
>3 |
|
10-5a |
>3 |
|
10-5b |
0 |
|
10-5c |
0 |
|
11-1c |
3 |
|
12-1b |
>3 |
|
12-1c |
>3 |
|
12-1d |
>3 |
|
12-2a |
0 |
|
13-1a |
>3 |
|
13-1b |
>3 |
|
14-1c |
>3 |
|
14-1e |
>3 |
|
15-2b |
>3 |
|
15-3a |
>3 |
|
15-3b |
>3 |
|
15-3d |
>3 |
|
16-2b |
0 |
|
16-3c |
>3 |
|
16-3d |
3 |
|
16-3e |
1 |
|
Religation Control |
0 |
Results:
|
96-well |
Positive Colonies green, negative Colonies red |
Red font: Maybe positive |
|||||||||||||||
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
||||||
|
1 |
1-2a |
1-2d |
3-4a 1 |
3-4a 2 |
3-4a 3 |
3-4b 1 |
3-4b 2 |
3-5b 1 |
3-5b 2 |
3-5b 3 |
|||||||
|
2 |
4-1a 1 |
4-1a 2 |
7-2b |
4-1b 1 |
4-1b 2 |
4-1b 3 |
5-2c |
6-1b 1 |
6-1b 2 |
6-1b 3 |
6-1c |
6-1d |
|||||
|
3 |
7-2a 1 |
7-2a 2 |
4-1a 3 |
7-2e |
8-1a 1 |
8-1a 2 |
8-1a 3 |
9-1a |
9-1b 1 |
9-1b 2 + 6-1b 3 |
9-1b 3 |
||||||
|
4 |
10-3a 1 |
10-3a 2 |
10-3a 3 |
10-3c 1 |
10-3c 2 |
10-3c 3 |
10-5a 1 |
10-5a 2 |
10-5a 3 |
11-1c 1 |
11-1c 2 |
11-1c 3 |
|||||
|
5 |
12-1b 1 |
12-1b 2 |
12-1b 3 |
12-1c 1 |
12-1c 2 |
12-1c 3 |
12-1d 1 |
12-1d 2 |
12-1d 3 |
13-1a 1 |
13-1a 2 |
13-1a 3 |
|||||
|
6 |
13-1b 1 |
13-1b 2 |
13-1b 3 |
14-1c 1 |
14-1c 2 |
14-1c 3 |
14-1e 1 |
14-1e 2 |
14-1e 3 |
15-2b 1 |
15-2b 2 |
15-2b 3 |
|||||
|
7 |
15-3a 1 |
15-3a 2 |
15-3a 3 |
15-3b 1 |
15-3b 2 |
15-3b 3 |
15-3d 1 |
15-3d 2 |
15-3d 3 |
16-3c 1 |
16-3c 2 |
16-3c 3 |
|||||
|
8 |
16-3e |
16-3d 1 |
16-3d 2 |
16-3d 3 |
|||||||||||||
▼2015-08-13 Copper Click – Test of Labeled Part 1 and Splinted ligation
To check if the azide-modified NTPs are incorporated the reactive azide is clicked to an alkyne activated FAM-alkyne fluorophore under copper catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC). Samples are after the reaction separated on a 20 % denaturing PAGE and visualized first in the fluorescent channel for FAM and afterwards stained with SYBR
Gold to visualize the RNA. Controls were performed as well.
Reaction conditions:
|
cStock |
cFinal |
V[µL] |
|
|
Phosphate buffer pH 7.0 |
100 mM |
50 mM |
12,5 |
|
FAM alkyne |
10 µM |
400 nM |
1 |
|
Azide modified RNA |
1 µM |
200 nM |
5 |
|
Cu(II)SO4 |
2 mM |
100 µM |
1.25 |
|
THPTA |
5 mM |
500 µM |
2.5 |
|
Sodium ascorbate |
10 mM |
1 mM |
2.5 |
|
H2O |
Ad 25 |
0.25 |
|
|
Final |
|
25 |
Conditions as Winz, 2012.
- All compounds were mixed in the given order
- Cu(II)SO4 and THPTA and H2O were mixed before adding to the mixture to let THPTA chelat the Cu
- Lastly sodium ascorbate was added to reduce the Cu(II) to Cu(I)
- Reaction was incubated for 1 h at 37 °C and afterwards put at -20 °C
Checking success on a 20 % denaturing PAGE
- Samples were mixed with an appropriate amount of 2 x loading dye and separated on a 20 % denaturing PAGE
- Gel was scanned with a Biorad ChemDoc, GE in fluorescent mode using the pre-set FAM parameters
- Afterwards gel was stained using SYBR gold and scanned with the Typoon in SYBR Gold mode
Result and Outlook:
Labeling of RNA with azide and splinted ligation was only partially successful. Labeling and splinted ligation was repeated with different conditions.
week number 32
▼2015-08-03 Digest – Ribozymes (Sequencing) (3.8.15)
|
|
Volume for 30µl [µl] |
|
DNA |
25 |
|
CutSmart |
2,5 |
|
Bmt |
0,5 |
|
BamHI –HF |
0,5 |
|
ddH2O |
1,5 |
- 5µl 2Log-Ladder (NEB)
- 5µl Purple Loading Dye (NEB)
▼2015-08-04 Ribozyme 1 _CFTR 1 T_ Digest and Ligation
j PCR, PCR Purification
k Digest, Gel
l Ligation
m Transformation
Digest Rib 1A, B
|
|
Volume for 30µl [µl] |
|
DNA |
25 |
|
CutSmart |
2,5 |
|
Bmt |
0,5 |
|
BamHI –HF |
0,5 |
|
ddH2O |
1,5 |
Ligation 1A, B
|
Ribozyme |
[ng/µl] |
[µl] |
|
1A |
20,4 |
0,20 |
|
1B |
13,4 |
0,30 |
|
|
Concentration [µl] |
|
|
Backbone |
2 |
|
|
Insert |
4ng |
|
|
T4- Ligase |
1 |
|
|
Buffer |
1 |
|
|
ddH2O |
5,8 (Rib 1A) |
5,7 (Rib 1B) |
▼2015-08-06 Insert cloning into the pSB1C3 Vector with MCS, pcat and Ribozyme
Procedures:
Digestion:
Description:
Steps:
- Set up reaction according to protocol:
ddH2O for a final volume of 20 µl
2 µl of 10x Reaction Buffer (e.g. NEB CutSmart)
0.5 µl of selected Enzyme(s)
ca. 1 µl of mini prep DNA (Range 200-1000 ng)
- Incubate at 37°C for 60'
The digestion was made in two steps in order to trigger the exonuclease activity of one of the endonucleases. The cutting sites of BglII and SalI are to narrow to each other for a endonuclease activity.
Step 1: Digestion with BglII
Components:
1 µg of Vector DNA
2 µl Cutsmart
1 µl of BglII
ad 20 µl ddH2O
Step 2: Digestion with SalI, triggering of SalI's exonuclease activity
Components:
8 µl of SalI-HF
10 µl of Cutsmart
52µl ddH2O
30 µl DNA
Aterwards a dephosphorylation and a PCR purification was made
Step 1: BglII digestion:
|
Nummer |
c |
µl/µg |
µl ddH20 |
|
7-2 |
224 |
5 |
12,5 |
|
8-1 |
210 |
5 |
12,5 |
|
8-2 |
519 |
2 |
15,5 |
|
8-3 |
471 |
3 |
14,5 |
|
9-1 |
205 |
5 |
12,5 |
|
9-2 |
507 |
2 |
15,5 |
|
9-5 |
448 |
3 |
14,5 |
|
10-1 |
226 |
5 |
12,5 |
|
10-2 |
220 |
5 |
12,5 |
|
10-3 |
369 |
3 |
14,5 |
|
10-5 |
542 |
2 |
15,5 |
|
11-1 |
307 |
4 |
13,5 |
|
12-1 |
534 |
2 |
15,5 |
|
12-2 |
106 |
10 |
7,5 |
|
12-4 |
499 |
3 |
14,5 |
|
13-1 |
221 |
5 |
12,5 |
|
14-1 |
542 |
2 |
15,5 |
|
14-3 |
303 |
4 |
13,5 |
|
15-2 |
407 |
3 |
14,5 |
|
15-3 |
317 |
4 |
13,5 |
|
3-4 |
130 |
8 |
9,5 |
|
3-5 |
120 |
9 |
8,5 |
|
4-1 |
129 |
8 |
9,5 |
|
5-2 |
64 |
16 |
1,5 |
|
6-1 |
89 |
12 |
5,5 |
|
16-3 |
85 |
12 |
5,5 |
Ligation
Description:
Materials and chemicals:
2 µl 10x T4 Ligase Buffer: thawed and resuspended at room temperature
1 parts Vector DNA (mol not l)
3 part Insert DNA (mol not l)
20 µl Nuclease free water
1 µl T4 DNA Ligase
Steps:
- Set up the reaction in a microcentrifuge tube on ice. T4 DNA Ligase should be added last. The molar ratio of vector to insert should be 1:3.
- Gently mix the reaction by pipetting up and down and microfuge briefly.
- For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours. Alternatively a high concentration of T4 Ligase can be used in a 10 minute ligation.
- Heat inactivate at 80°C for 10 minutes
- Chill on ice and transform 1-5 µl of the reaction into 50 µl of competent cells.
Chemicals:
25 ng cutted Vector DNA
8 ng cutted Insert DNA
2 µl DNA-Ligase Buffer
1 µl DNA-Ligase
ad 20 µl ddH2O
|
Nr |
Ribozym |
c |
Insert Nr |
µl Insert |
µl Vector |
µl ddH20 |
T4-Ligase-Buffer |
T4-Ligase |
|
3-4 |
CFTR 1 C |
8,5 |
C1V |
0,2 |
3 |
13,8 |
2 |
1 |
|
3-5 |
CFTR 1 C |
16,3 |
C1V |
0,2 |
2 |
14,8 |
2 |
1 |
|
4-1 |
CFTR 2 T |
15,2 |
C2V |
0,2 |
2 |
14,8 |
2 |
1 |
|
5-2 |
CFTR 2 A |
15,2 |
C2V |
0,2 |
2 |
14,8 |
2 |
1 |
|
6-1 |
CFTR 2 C |
21,4 |
C2V |
0,2 |
2 |
14,8 |
2 |
1 |
|
7-2 |
CFTR 1 DE T |
7,2 |
C1V |
0,2 |
4 |
12,8 |
2 |
1 |
|
8-1 |
CFTR 1 DE A |
3,8 |
C1V |
0,2 |
7 |
9,8 |
2 |
1 |
|
8-2 |
CFTR 1 DE A |
10,7 |
C1V |
0,2 |
3 |
13,8 |
2 |
1 |
|
8-3 |
CFTR 1 DE A |
16,6 |
C1V |
0,2 |
2 |
14,8 |
2 |
1 |
|
9-1 |
CFTR 1 DE C |
4,2 |
C1V |
0,2 |
6 |
10,8 |
2 |
1 |
|
9-2 |
CFTR 1 DE C |
5,7 |
C1V |
0,2 |
5 |
11,8 |
2 |
1 |
|
9-5 |
CFTR 1 DE C |
8,9 |
C1V |
0,2 |
3 |
13,8 |
2 |
1 |
|
10-1 |
CFTR 2 DE T |
5 |
C2V |
0,2 |
5 |
11,8 |
2 |
1 |
|
10-2 |
CFTR 2 DE T |
5,7 |
C2V |
0,2 |
5 |
11,8 |
2 |
1 |
|
10-3 |
CFTR 2 DE T |
10 |
C2V |
0,2 |
3 |
13,8 |
2 |
1 |
|
10-5 |
CFTR 2 DE T |
10,5 |
C2V |
0,2 |
3 |
13,8 |
2 |
1 |
|
11-1 |
CFTR 2 DE a |
15 |
C2V |
0,2 |
2 |
14,8 |
2 |
1 |
|
12-1 |
CFTR 2 DE C |
13,6 |
C2V |
0,2 |
2 |
14,8 |
2 |
1 |
|
12-2 |
CFTR 2 DE C |
9 |
C2V |
0,2 |
3 |
13,8 |
2 |
1 |
|
13-1 |
GFP 1 DE |
2,9 |
G1V |
0,2 |
9 |
7,8 |
2 |
1 |
|
14-1 |
GFP 2 DE |
17,6 |
G2V |
0,2 |
2 |
14,8 |
2 |
1 |
|
14-3 |
GFP 2 DE |
6 |
G2V |
0,2 |
5 |
11,8 |
2 |
1 |
|
15-2 |
GFP 1 |
14,3 |
G1V |
0,2 |
2 |
14,8 |
2 |
1 |
|
15-3 |
GFP 1 |
12,1 |
G1V |
0,2 |
3 |
13,8 |
2 |
1 |
|
16-3 |
GFP 2 |
14 |
G2V |
0,2 |
2 |
14,8 |
2 |
1 |
|
Insert Nr |
c |
|
C1V |
49,8 |
|
C2V |
41,4 |
|
G1V |
46,6 |
|
G2V |
40,6 |
KCM Transformation:
Description:
Steps:
- Take 50µl chemical competent E. coli from -80 freezer and thaw on ice
- Add (as master mix):
2,5µl DNA
10µl KCM 5x
37,5µl H2O
- Incubate on ice for 30 minutes
- Heat shock at 42°C for 1 minute
- Incubate on ice for 2 minutes
- Add 900 µl of LB or 2x YT Medium
- Incubate on 37°C for 60min
- Centrifuge 5min at 1000g
- Take 900µl of supernatant and throw away
- Resuspend pellet in remaining media
- Plate out on agar with antibiotics (1:1 / 1:10)
Colony PCR:
Description:
Materials and Chemicals:
PCR-tubes
Forward primer
Reverse primer
Masermix (dNTPs, Polymerase, buffer)
Water
Thermocycler
Endvolume: 10 µl
Steps:
- Pick colonies from plates. Solute one colony in about 20 µl of water.
- Give the colonies into 10 µl colony PCR solution with OneTaq Mastermix (which should be diluted to 1x in the end (e.g. you need 5 µl of 2x mastermix for 10 µl)) and primer (between 0,1 and 1 µl)
Use the Thermocycler with an appropriate PCR program for at least 25 cycles
Results:
The colony PCR showed at least one positive clone for every ribozyme.
Results:
The plates gave a high number of clones. Out of them either new plates were made or single colonies were found, that were given into a colony PCR. Positive Ribozyme colonies were minipreped and gave a yield of 150-325 ng/µl DNA.
First colony PCR: (Clones are named: x-y A: x = 1-15 (number of ribozyme), y = number of the colony, A: Colony capital letter (A-E))
|
Colony ID |
Positve/Negative/Multiple Bands |
|
15-3 A |
Pos |
|
15-3 B |
Pos |
|
15-3 C |
Neg |
|
15-3 D |
Pos |
|
14-3 A |
Neg |
|
14-3 B |
Neg |
|
12-1 A |
Pos |
|
12-1 B |
Pos |
|
12-1 C |
Pos |
|
12-1 D |
Pos |
|
12-1 E |
Neg |
|
10-3 A |
Double Band |
|
10-3 B |
Neg |
|
10-3 C |
Pos |
|
10-2 A |
Pos |
|
10-2 B |
Pos |
|
10-2 C |
Neg |
|
9-1 A |
Double Band |
|
9-1 B |
Double Band |
|
9-1 C |
Pos |
|
8-1 A |
Pos |
|
8-1 B |
Neg |
|
6-1 A |
Neg |
|
6-1 B |
Pos |
|
6-1 C |
Pos |
|
6-1 D |
Pos |
|
6-1 E |
Neg |
|
4-1 A |
Pos |
|
4-1 B |
Pos |
|
10-5 A |
Pos |
|
10-5 B |
Pos |
|
10-5 C |
Double Band |
|
10-5 D |
Neg |
|
10-5 E |
Neg |
|
11-1 A |
Neg |
|
11-1 B |
Neg |
|
11-1 C |
neg |
|
13-1 A |
Pos |
|
13-1 B |
Pos |
|
15-2 A |
Neg |
|
15-2 B |
Triple Band |
|
15-2 C |
Neg |
week number 31
▼2015-07-27 PCR amplification of the ribozymes CFTR2 A/T/C for in vitro transcription
Procedures:
Amplification PCR 1
Description
Chemicals:
5 µl Q5 Master Mix
0,5 µl Ribozymes from stock solution
1 µl Primer fwd
1 µl Primer rev
2,5 µl ddH2O
Program:
98°C for 1 minute
------------------------------
98°C for 10 seconds
60°C for 20 seconds
72°C for 30 seconds
Repeat 35 times
------------------------------
72°C for 2 minutes
------------------------------
4°C for holding
Results:
After recognizing the success of the first PCR on a gel, a second PCR was made with to get more DNA.
Amplification PCR 2:
Description:
Chemicals:
25 µl Q5 Master Mix
0,5 µl Ribozymes from prior PCR reaction
5 µl Primer fwd
5 µl Primer rev
14,5 µl ddH2O
Program:
98°C for 30 seconds
------------------------------
98°C for 10 seconds
60°C for 15 seconds
72°C for 20 seconds
Repeat 35 times
------------------------------
72°C for 1 minutes
------------------------------
4°C for holding
▼2015-07-28 Digest-Ribozyme amplification
|
|
Volume [µl] |
|
DNA |
15 |
|
CutSmart |
3 |
|
BamHI-HF |
0.3 |
|
BmtI-HF |
0.3 |
|
ddH2O |
11.4 |
Conditions
- Duration: 1h
- Temperature: 37°C
- 350 rpm
Concentration
|
Ribozyme |
Concentration [ng/µl] |
|
1 CFTR 1 T |
8,9 |
|
2 CFTR 1 A |
15,9 |
|
3 CFTR 1 C |
22,9 |
|
4 CFTR 2 T |
1,5 |
|
5 CFTR 2 A |
15,6 |
|
6 CFTR 2 C |
31,9 |
|
15 GFP 1 |
14,2 |
|
16 GFP 2 |
17,8 |
- PCR Purification Kit
▼2015-07-28 Ligation (28.07.15)
|
|
Volume [µl] |
|
Backbone |
10ng -> 2 |
|
Insert |
4ng |
|
T4-Ligase |
1 |
|
Buffer |
1 |
|
ddH2O |
to 20µl |
Conditions
- Duration: 15min
- Temperature: 25°C
- 350 rpm
▼2015-07-28 KCM - Transformation
See also LabGuru protocol
▼2015-07-29 Picking colonies
Picked colonies
|
Ribozyme |
Colony1 |
Colony 2 |
Colony 3 |
Colony 4 |
Colony 5 |
|
1 CFTR 1 T |
/ |
/ |
/ |
/ |
/ |
|
2 CFTR 1 A |
2-1 |
2-2 |
2-3 |
2-4 |
2-5 |
|
3 CFTR 1 C |
3-1 |
3-2 |
3-3 |
3-4 |
3-5 |
|
4 CFTR 2 T |
4-1 |
4-2 |
4-3 |
4-4 |
4-5 |
|
5 CFTR 2 A |
5-1 |
5-2 |
5-3 |
5-4 |
5-5 |
|
6 CFTR 2 C |
6-1 |
6-2 |
6-3 |
6-4 |
6-5 |
|
15 GFP 1 |
15-1 |
15-2 |
15-3 |
15-4 |
15-5 |
|
16 GFP 2 |
16-1 |
16-2 |
16-3 |
16-4 |
16-5 |
▼2015-07-29 colony PCR
Colony PCR
|
|
Volume [ng/µl] |
|
Primer fwd |
0,5 |
|
Primer rvs |
0,5 |
|
OneTaq Polymerase |
5 |
|
ddH2O |
4 |
▼2015-07-30 Repetition Ribozyme 1 and 15
PCR (GEL image)
Digest (GEL image)
|
Ribozyme |
Concentration [ng/µl] |
|
1 CFTR 1 T |
17,4 |
|
15 GFP 1 |
6 |
|
Backbone |
37 |
Ligation
|
ddH2O |
To 20µl |
|
Backbone |
50ng |
|
Insert |
20ng |
|
T4 Ligase |
1µl |
|
Buffer |
2µl |
Transformation
▼2015-07-30 Mutation correction PCR of p415-GPD
Description:
In order to correct a stop-codon mutation in the CFTR-testconstruct a mutation correction PCR was made.
Mutation correction PCR:
Description:
Chemicals:
0,2 µl p415-GPD CFTR-test
1 µl Primer fwd
1 µl Primer rev
2,8 µl ddH2O
5 µl Q5 Master Mix
Program:
98°C for 2 minutes
--------------------------
98°C for 30 seconds
Annealing temperature for 30 seconds 35 cycles
72°C for 4 minutes
--------------------------
72°C for 5 minutes
--------------------------
4°C for holding
The reaction was set in a triplet with 3 different annealing temperatures: 72°C (2step), 70°C and 68°C
DpnI digestion:
Description:
1 µl of DnpI was given on the PCR mix after the PCR reaction. The mix was incubated at 37°C for 4 hours and then inactivated at 65°C for 20 minutes.
The DNA was given on a gel after the DpnI-digestion
Results:
The gel picture showed just smear.
▼2015-07-31 Miniprep – QIAGEN Kit (31.7.15)_concentrations
|
Ribozyme + number |
Concentration [ng/µl] |
Volume for Sequencing(40ng) [µl] |
|
1 |
/ |
/ |
|
2_2-2 |
99 |
6,06 |
|
3_3-4/5 |
90,6 |
6,62 |
|
4_4-1/4-2 |
116,9 |
5,13 |
|
5_5-1/2 |
99,3 |
6,04 |
|
6_6-1 |
87,3 |
6,87 |
|
15 |
|
|
|
16_16-3 |
89,8 |
6,68 |
▼2015-07-31 Colony PCR and mini prep of Ribozymes
Colony PCR
Miniprep – QIAGEN Kit
|
Concentration [ng/µl] |
Volume for Sequencing(40ng) [µl] |
|
|
1 |
/ |
/ |
|
2_2-2 |
99 |
6,06 |
|
3_3-4/5 |
90,6 |
6,62 |
|
4_4-1/4-2 |
116,9 |
5,13 |
|
5_5-1/2 |
99,3 |
6,04 |
|
6_6-1 |
87,3 |
6,87 |
|
15 |
|
|
|
16_16-3 |
89,8 |
6,68 |
▼2015-07-31 Repairing the deletion in the HDV region of the twin ribozymes with assembly PCR + Cloning
Description:
In order to repair a deletion in the DE inserts and ribozymes, 3 PCRs were made. In the first PCR the parts were elongated with a Primer overhang that carries the DNA fragment to fill up the deletion. The second was an assembly PCR. The third has been done to amplify the fragments.
|
ID # |
Ribozyme |
|
1 |
CFTR 1 T |
|
2 |
CFTR 1 A |
|
3 |
CFTR 1 C |
|
4 |
CFTR 2 T |
|
5 |
CFTR 2 A |
|
6 |
CFTR 2 C |
|
7 |
CFTR 1 DE T |
|
8 |
CFTR 1 DE A |
|
9 |
CFTR 1 DE C |
|
10 |
CFTR 2 DE T |
|
11 |
CFTR 2 DE A |
|
12 |
CFTR 2 DE C |
|
13 |
GFP 1 DE |
|
14 |
GFP 2 DE |
|
15 |
GFP 1 |
|
16 |
GFP 2 |
PCR: Division and Elongation
Description
PCR-Mix:
5 µl Primer fwd
5 µl Primer rev
0,5 µl Template
14,5 µl ddH2O
25 µl Q5-MasterMix
Program:
98°C for 30 seconds
------------------------------------------------
98°C for 10 seconds
68°C for 10 seconds 35 Cycles
72°C for 15 seconds
------------------------------------------------
72°C for 30 seconds
------------------------------------------------
4°C for holding
The PCR to make two parts is made in two different tubes, one for the first and one for the second part.
First part:
|
Ribozyme/Insert |
Fwd Primer |
Rev Primer |
|
CFTR 1 DE T |
DH_39 |
MJ_15 |
|
CFTR 1 DE A |
DH_39 |
MJ_15 |
|
CFTR 1 DE C |
DH_39 |
MJ_15 |
|
CFTR 2 DE T |
DH_40 |
MJ_15 |
|
CFTR 2 DE A |
DH_40 |
MJ_15 |
|
CFTR 2 DE C |
DH_40 |
MJ_15 |
|
GFP 1 DE |
DH_41 |
MJ_18 |
|
GFP 2 DE |
DH_41 |
MJ_18 |
|
Insert CFTR 1 |
DH_18a |
MJ_17 |
|
Insert CFTR 2 |
DH_18a |
MJ_16 |
|
Insert GFP 1 |
DH_20a |
MJ_20 |
|
Insert GFP 2 |
DH_22a |
MJ_19 |
Second Part:
|
Riobzyme/Insert |
Fwd-Primer |
Rev-Primer |
Tubes |
|
CFTR 1 DE T |
MJ_21 |
DH_48 |
4 |
|
Insert CFTR 1 |
MJ_21 |
DH_27a |
2 |
Assembly PCR
Description:
PCR-Mix:
1:1 Molar ratio of part 1 and part 2 (from the first PCR)
25 µl Q5-MasterMix
ad 40 µl ddH2O
PCR-program:
98°C for 1 minute
--------------------------------------------
98°C for 10 seconds
70°C with a ramp rate of 1°C per second for 30 seconds
72°C for 30 seconds
Repeat 3 Times
--------------------------------------------
72°C for 30 seconds
-------------------------------------------
4°C for holding
|
Frag-ment 1 |
Length |
ng/µl |
nmol |
Frag-ment 2 |
Length |
ng/µl |
nmol |
Frag-ment 2 Ratio |
F1 µl |
F2 µl |
ddH2O µl |
|
|
2T |
226 |
142 |
1,017 |
Rib |
80 |
327,2 |
6,62 |
0,15 |
1,5 |
0,2 |
23,3 |
|
|
2A |
226 |
184,9 |
1,324 |
Rib |
80 |
327,2 |
6,62 |
0,2 |
1,5 |
0,3 |
23,2 |
|
|
2C |
226 |
117,5 |
0,841 |
Rib |
80 |
327,2 |
6,62 |
0,12 |
1,5 |
0,2 |
23,3 |
|
|
G1 |
227 |
199,3 |
1,42 |
Rib |
80 |
327,2 |
6,62 |
0,2 |
1,5 |
0,3 |
23,2 |
|
|
G2 |
243 |
166,5 |
1,109 |
Rib |
80 |
327,2 |
6,62 |
0,16 |
1,5 |
0,3 |
23,2 |
|
|
IC1 |
109 |
120,2 |
1,784 |
Ins |
87 |
205,6 |
6,62 |
0,46 |
1,5 |
0,7 |
22,8 |
|
|
IC2 |
118 |
128,3 |
1,759 |
Ins |
87 |
205,6 |
6,62 |
0,46 |
1,5 |
0,7 |
22,8 |
|
|
1T |
216 |
150,2 |
1,125 |
Rib |
80 |
327,2 |
6,62 |
0,17 |
1,5 |
0,3 |
23,2 |
|
|
1A |
216 |
121 |
0,906 |
Rib |
80 |
327,2 |
6,62 |
0,14 |
1,5 |
0,2 |
23,3 |
|
|
1C |
216 |
98,5 |
0,737 |
Rib |
80 |
327,2 |
6,62 |
0,11 |
1,5 |
0,2 |
23,3 |
|
|
IG1 |
114 |
145,7 |
2,067 |
Ins |
87 |
205,6 |
6,62 |
0,54 |
1,5 |
0,8 |
22,7 |
|
|
IG2 |
115 |
151,3 |
2,128 |
Ins |
87 |
205,6 |
6,62 |
0,56 |
1,5 |
0,8 |
22,7 |
|
PCR: Amplification
Description:
PCR-Mix:
5 µl Primer fwd and rev are given into the assembly PCR mix after the assembly PCR
Program:
98°C for 60 seconds
----------------------------------
98°C for 10 seconds
68°C for 15 seconds 35 Cycles
72°C for 20 seconds
------------------------------------
72°C for 30 seconds
------------------------------------
4°C for holding
Digestion:
Description:
Steps:
- Set up reaction according to protocol:
ddH2O for a final volume of 20 µl
2 µl of 10x Reaction Buffer (e.g. NEB CutSmart)
0.5 µl of selected Enzyme(s)
Ca. 1 µl of mini prep DNA (Range 200-1000 ng)
- Incubate at 37°C for 60'
Digestion of the ribozymes was made with BamHI and BmtI.
Ligation
Description:
Materials and chemicals:
2 µl 10x T4 Ligase Buffer: thawed and resuspended at room temperature
1 parts Vector DNA (mol not l)
3 part Insert DNA (mol not l)
20 µl Nuclease free water
1 µl T4 DNA Ligase
Steps:
- Set up the reaction in a microcentrifuge tube on ice. T4 DNA Ligase should be added last. The molar ratio of vector to insert should be 1:3.
- Gently mix the reaction by pipetting up and down and microfuge briefly.
- For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
- For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours. Alternatively a high concentration of T4 Ligase can be used in a 10 minute ligation.
- Heat inactivate at 80°C for 10 minutes
- Chill on ice and transform 1-5 µl of the reaction into 50 µl of competent cells.
The ribozymes were ligated into a cutted and dephoshorylated pSB1C3 with MCS and pcat.
KCM Transformation:
Description:
Steps:
- Take 50µl chemical competent E. coli from -80 freezer and thaw on ice
- Add (as master mix):
2,5µl DNA
10µl KCM 5x
37,5µl H2O
- Incubate on ice for 30 minutes
- Heat shock at 42°C for 1 minute
- Incubate on ice for 2 minutes
- Add 900 µl of LB or 2x YT Medium
- Incubate on 37°C for 60min
- Centrifuge 5min at 1000g
- Take 900µl of supernatant and throw away
- Resuspend pellet in remaining media
- Plate out on agar with antibiotics (1:1 / 1:10)
Colony PCR:
Description:
Materials and Chemicals:
PCR-tubes
Forward primer
Reverse primer
Masermix (dNTPs, Polymerase, buffer)
Water
Thermocycler
Endvolume: 10 µl
Steps:
- Pick colonies from plates. Solute one colony in about 20 µl of water.
- Give the colonies into 10 µl colony PCR solution with OneTaq Mastermix (which should be diluted to 1x in the end (e.g. you need 5 µl of 2x mastermix for 10 µl)) and primer (between 0,1 and 1 µl)
Use the Thermocycler with an appropriate PCR program for at least 25 cycles
Results:
The colony PCR showed at least one positive clone for every ribozyme
Positive Colonies: 7-2, 8-1, 8-2, 8-3, 9-1, 9-2, 9-5, 10-1, 10-2, 10-3, 10-5, 11-1, 12-1, 12-2
Positive Colonies: 12-4, 13-1, 14-1, 14-3, 15-2, 15-3
week number 30
▼2015-07-24 Yeast transformation with p415-GPD with CFTR-testconstruct without mutation correction
Description
Yeast was transformated with p415-GPD with the CFTR-testconstruct from a miniprep of an E.coli culture. Transformed cells should turn red.
Procedures:
Yeast transformation 24.07
Description:
Materials and chemicals:
10 µl of cells for transformation with a plasmid, 50 µl of cells for transformation with a PCR product
2 µl of plasmid DNA per 10 µl of cells (200 ng of DNA)
6 equivalents of PEG to the yeast cells
1/9 of DMSO to the volume of plasmids, yeast cells and PEG
100 - 200 µl of liquid medium
Steps:
- Give the plasmid DNA into a sterile 1.5 ml tube (2 µl per 10 µl of cells. Add the thawed competent cells.
- Mix the suspension well, then add the PEG.
- Incubate for 30 mins at room temperature while mixing (the cells can be incubated up to 2 hours).
- Add the DMSO to get a final concentration of 10% DMSO.
- Place the yeast in a 42°C water bath for 5-20 minutes. NO Thermomixer.
- Centrifuge cells for 2-3 minutes at 2000 rpm/500g
- Discard the supernatant and resuspend the yeast in the liqiud YPD medium
The transformation was repeated on 27.07.2015
Results:
Transformation 1:
The first transformation resulted in cultures with an ununsual behaivior, maybe the plates were contaminated with another organism. The cells didn't turned red
Transformation 2:
A test run on a FACS showed that the transformed cells are functional and express the CFTR-testconstruct
▼2015-07-25 Mutation correction PCR p415-GPD with CFTR-testconstruct
Description
In order to correct the mutation in the mCherry part of the CFTR-testcontruct, a mutation PCR was made.
Procedures:
Mutation correction PCR 1:
Description
Chemicals:
0,1 µl p415-GPD with CFTR-testconstruct (about 650 ng/µl)
1 µl Primer fwd
1 µl Primer rev
2,9 µl ddH2O
5 µl Q5
Program:
2-step PCR:
98°C
-----------------------
98°C
72°C
------------------------
72°C
------------------------
4°C for holding
Mutation correction PCR 2
Description:
Chemicals:
0,1 µl p415-GPD with CFTR-testcontruct
1 µl Primer fwd
1 µl Primer rev
2,9 µl ddH2O
5 µl Q5
Program:
98°C for 1 minute
------------------------
98°C for 20 s
Anneal for 20 s
72°C for 4 minutes
Repeat cycle 35 times
-------------------------
72°C for 5 minutes
-------------------------
4°C for holding
Tried annealing temperatures:
72°C (2-step-PCR)
70,5°C
68,8°C
65,6°C
64°C
Annealing temperatures were made by gradient
Results:
Mutation PCR 1:
The Gel showed there was just smear
Mutation PCR 2:
Every Annealing temperature resulted in smear and no detectable fragment
week number 29
▼2015-07-14 Inserting twin ribozymes into pSB1C3 + MCS + pcat
Description:
After validating the MCS + pcat fragment, the ribozymes were cloned into pSB1C3
Procedures:
2-step digest:
Description:
Steps:
- Set up reaction according to protocol:
ddH2O for a final volume of 20 µl
2 µl of 10x Reaction Buffer (e.g. NEB CutSmart)
0.5 µl of selected Enzyme(s)
ca. 1 µl of mini prep DNA (Range 200-1000 ng)
- Incubate at 37°C for 60'
- Load on gel (add loading dye first)
Two digestion steps were made:
Step 1:
- Set up following mix:
25 µl pSB1C3-MCS-pcat
1 µl BmtI
5 µl CutSmart 10x Buffer
19 µl ddH2O
- Digestion for 1 hour at 37°C
- PCR-purification with 2 step elution in 35 µl water
Step 2:
- Set up following mix:
35 µl from the PCR-purification
10 µl BamHF
5 µl CutSmart
- Digestion for 4 hours at 37°C
- PCR purification with 2 step elution in 30 µl Elution Buffer
Ligation:
Description:
Materials and chemicals:
2 µl 10x T4 Ligase Buffer: thawed and resuspended at room temperature
1 parts Vector DNA (mol not l)
3 part Insert DNA (mol not l)
20 µl Nuclease free water
1 µl T4 DNA Ligase
Steps:
- Set up the reaction in a microcentrifuge tube on ice. T4 DNA Ligase should be added last. The molar ratio of vector to insert should be 1:3.
- Gently mix the reaction by pipetting up and down and microfuge briefly.
- For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
- For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours. Alternatively a high concentration of T4 Ligase can be used in a 10 minute ligation.
- Heat inactivate at 80°C for 10 minutes
- Chill on ice and transform 1-5 µl of the reaction into 50 µl of competent cells.
We made one ligation for every ribozyme and one as a religation control (17 in total)
Ligation mixes:
Not DE-ribozymes (without Hammerhead and Hepatitis Delta Virus ribozyme on the ends):
1 µl Backbone (35 ng)
2,5 µl Insert (12,5 ng)
1 µl T4-Ligase
2 µl T4-Ligation Buffer 10x
13,5 µl ddH2O
DE-ribozymes (with HH and HDV):
1 µl Backbone
4 µl Insert
1 µl T4-Ligase
2 µl T4-Ligation Buffer 10x
12 µl ddH2O
Religation Control:
1 µl Backbone
1 µl T4-Ligase
2 µl T4-Ligation Buffer 10x
16 µl ddH2O
KCM Transformation:
Description:
Steps:
- Take 50µl chemical competent E. coli from -80 freezer and thaw on ice
- Add (as master mix):
2,5µl DNA
10µl KCM 5x
37,5µl H2O
- Incubate on ice for 30 minutes
- Heat shock at 42°C for 1 minute
- Incubate on ice for 2 minutes
- Add 900 µl of LB or 2x YT Medium
- Incubate on 37°C for 60min
- Centrifuge 5min at 1000g
- Take 900µl of supernatant and throw away
- Resuspend pellet in remaining media
- Plate out on agar with antibiotics (1:1 / 1:10)
We made 1 transformation for every ligation (17 in total), one for each ribozyme and one as a religation control
Colony PCR:
Description:
Materials and Chemicals:
PCR-tubes
Forward primer
Reverse primer
Masermix (dNTPs, Polymerase, buffer)
Water
Thermocycler
Endvolume: 10 µl
Steps:
- Pick colonies from plates. Solute one colony in about 20 µl of water.
- Give the colonies into 10 µl colony PCR solution with OneTaq Mastermix (which should be diluted to 1x in the end (e.g. you need 5 µl of 2x mastermix for 10 µl)) and primer (between 0,1 and 1 µl)
- Use the Thermocycler with an appropriate PCR program for at least 25 cycles
We picked 12 colonies from every ribozyme plate and got no satisfying results
▼2015-07-15 Digestion of pSB1C3-MCS-pcat with BmtI and BamHI 2 step
Description:
We made a 2 step digestion with BmtI and BamHI for 1 and 4 hours to proper cut the pSB1C3 in the MCS region, because the BmtI and BamHI cutting sites are next to each other and the exonuclease activity of the BamHI needs to be triggered.
Procedures:
2 step ligation:
Description:
First digetion with BmtI:
Steps:
- Make a mix of following chemicals in a 1,5 ml centrifuge tube:
25 µl pSB1C3-MCS-pcat
1 µl BmtI
5 µl CutSmart 10x Buffer
19 µl ddH2O
- Incubate for 1 hour at 37°C
- Make a PCR purification
Second digestion with BamHI:
Steps:
- Make a mix of following chemicals:
35 µl of DNA solution from the prior PCR purification
10 µl BamHI
5 µl CutSmart 10x Buffer
- Incubate for 4 hours at 37°C
- Make a PCR purification
Results:
Nanodrop result: 24,6 ng/µl
▼2015-07-15 Digestion of the ribozyme fragments
Description:
The ribozymes were cut for the insertion into pSB1C3 with MCS/pcat.
Procedures:
Ribozyme digestion:
Description:
Steps
- Chemicals (per tube):
6 µl DNA solution (~60 ng)
1 µl Cutsmart
0,1 µl BamHI
01, µl BmtI
2,8 µl ddH2O
- Incubation for 1 hour at 37°C
- PCR-purificaiton, with 2 step-elution
- Lyophylisation
- Solute dried DNA in 10 µl water
Results:
Nanodrop results:
|
Number |
Ribozyme |
Nanodrop-Result |
|
1 |
CFTR 1 T |
19,4 |
|
2 |
CFTR 1 A |
77 |
|
3 |
CFTR 1 C |
94,8 |
|
4 |
CFTR 2 A |
97 |
|
5 |
CFTR 2 C |
91,1 |
|
6 |
GFP 1 |
77,3 |
|
7 |
GFP 1 DE |
38,6 |
|
8 |
CFTR 2 DE C |
55,3 |
|
9 |
CFTR 1 DE T |
72,7 |
|
10 |
CFTR 1 DE A |
60,6 |
|
11 |
GFP 2 DE |
65,6 |
|
12 |
CFTR 1 DE C |
56,3 |
|
13 |
CFTR 2 DE T |
100,2 |
|
14 |
CFTR 1 DE A |
29,3 |
|
15 |
CFTR 2 T |
62,5 |
|
16 |
GFP 2 |
94,2 |
▼2015-07-15 Ligation of the cutted pSB1C3-MCS-pcat with ribozyme fragments
Procedures:
Ligation:
Description:
Materials and chemicals:
2 µl 10x T4 Ligase Buffer: thawed and resuspended at room temperature
1 parts Vector DNA (mol not l)
3 part Insert DNA (mol not l)
20 µl Nuclease free water
1 µl T4 DNA Ligase
Steps:
- Set up the reaction in a microcentrifuge tube on ice. T4 DNA Ligase should be added last. The molar ratio of vector to insert should be 1:3.
- Gently mix the reaction by pipetting up and down and microfuge briefly.
- For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
- For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours. Alternatively a high concentration of T4 Ligase can be used in a 10 minute ligation.
- Heat inactivate at 80°C for 10 minutes
- Chill on ice and transform 1-5 µl of the reaction into 50 µl of competent cells.
Pipetting sheet:
For fragment numbers see digestion of ribozyme fragments protocol
In one tube:
40 ng of pSB1C3 (about 1,5 µl)
13 ng of non-DE-ribozymes or 22 ng of DE ribozymes
1 µl Ligase
2 µl Buffer
Mastermix: pSB1C3, Ligase used 5x concentrated ligase (2 Million U per ml) diluted with ddH2O, Buffer
Everything in µl:
|
Fragment-Number |
Mastermix |
Insert |
ddH2O |
|
1 |
4,5 |
0,7 |
14,8 |
|
2 |
4,5 |
0,2 |
15,3 |
|
3 |
4,5 |
0,2 |
15,3 |
|
4 |
4,5 |
0,2 |
15,3 |
|
5 |
4,5 |
0,2 |
15,3 |
|
6 |
4,5 |
0,2 |
15,3 |
|
7 |
4,5 |
0,6 |
14,9 |
|
8 |
4,5 |
0,4 |
15,1 |
|
9 |
4,5 |
0,3 |
15,2 |
|
10 |
4,5 |
0,4 |
15,1 |
|
11 |
4,5 |
0,4 |
15,1 |
|
12 |
4,5 |
0,4 |
15,1 |
|
13 |
4,5 |
0,3 |
15,2 |
|
14 |
4,5 |
0,8 |
14,7 |
|
15 |
4,5 |
0,2 |
15,3 |
|
16 |
4,5 |
0,2 |
15,3 |
▼2015-07-16 Digestion of pSB1C3-MCS-pcat with BmtI and BamHI 2 step
Description:
We made a 2 step digestion with BmtI and BamHI for 1 and 4 hours to proper cut the pSB1C3 in the MCS region, because the BmtI and BamHI cutting sites are next to each other and the exonuclease activity of the BamHI needs to be triggered.
Procedures:
2 step ligation:
Description:
First digetion with BmtI:
Steps:
- Make a mix of following chemicals in a 1,5 ml centrifuge tube:
25 µl pSB1C3-MCS-pcat
1 µl BmtI
5 µl CutSmart 10x Buffer
19 µl ddH2O
- Incubate for 1 hour at 37°C
- Make a PCR purification
Second digestion with BamHI:
Steps:
- Make a mix of following chemicals:
35 µl of DNA solution from the prior PCR purification
10 µl BamHI
5 µl CutSmart 10x Buffer
- Incubate for 4 hours at 37°C
- Make a PCR purification
▼2015-07-18 Outamplification and transformation of the CFTR-testconstruct
Description:
In order to change the cloning system from Gibson to restriction cloning, the CFTR fragment has been first mutated (single base mutation on sequencing data) and then outamplified. Afterwards it has been cutted, ligated with the vector and then transformed into E. coli
Mutation PCR #1:
Description:
Materials and chemicals:
25 µl Phusion flash
1 µl Primer fwd Dh_23
1 µl Primer rev DH_24
0,5 µl p413+CFTR-construct (which is inserted the other way around)
22,5 µl water
PCR:
98°C for 1 min
--------------------
98°C for 10 sec
72°C for 2 minutes
(Repeat 34 times)
---------------------
72°C for 4 minutes
4°C for holding
Mutation PCR #2
Description:
Materials and chemicals:
5 µl Phusion flash
1 µl Primer fwd Dh_23
1 µl Primer rev DH_24
0,5 µl p413+CFTR-construct (which is inserted the other way around)
2,5 µl water
PCR:
98°C for 1 min
--------------------
98°C for 10 sec
Annealing temperature for 30 sec
72°C for 1,5 min
(Repeat 34 times)
---------------------
72°C for 4 minutes
4°C for holding
4 samples with different annealing temperatures were made:
1: 72°C
2: 70°C
3: 68°C
4: 65°C
Materials and chemicals:
5 µl Phusion flash
1 µl Primer fwd Dh_23
1 µl Primer rev DH_24
0,5 µl p413+CFTR-construct (which is inserted the other way around)
2,5 µl water
PCR:
98°C for 1 min
--------------------
98°C for 10 sec
Annealing temperature for 30 sec
72°C for 1,5 min
(Repeat 34 times)
---------------------
72°C for 4 minutes
4°C for holding
4 samples with different annealing temperatures were made:
1: 72°C
2: 70°C
3: 68°C
4: 65°C
Materials and chemicals:
5 µl Phusion flash
1 µl Primer fwd Dh_23
1 µl Primer rev DH_24
0,5 µl p413+CFTR-construct (which is inserted the other way around)
2,5 µl water
PCR:
98°C for 1 min
--------------------
98°C for 10 sec
Annealing temperature for 30 sec
72°C for 1,5 min
(Repeat 34 times)
---------------------
72°C for 4 minutes
4°C for holding
4 samples with different annealing temperatures were made:
1: 72°C
2: 70°C
3: 68°C
4: 65°C
The 65°C annealing temperature gave the best results and further work was made with the sample 4.
Outamplification and rebuild of the end sites for the CFTR construct
Description:
Materials and chemicals:
25 µl Phusion Flash
1 µl DH_25
1 µl DH_26
1 µl DNA from Sample 4 of mutation PCR #2
22 µl ddH2O
PCR-program:
98°C for 1 min
------------------------
98°C for 10 s
64°C for 15 s
72°C for 20 s
(Repeat 34 times)
-----------------------
72°C for 1 min
4°C for holding
CFTR-Digestion:
Digestion CFTR-construct:
26 µl CFTR-Test construct
5 µl BamHI
5 µl HindIII
4 µl CutSmart 10x
Digest 3 hours at 37°C
Digestion p413-GPD:
10 µl p413-GPD
1 µl BamHI
1 µl HindIII
2 µl CutSmart 10x
6 µl ddH2O
Digest 60 minutes at 37°C
Ligation:
Description:
Materials and chemicals:
2 µl 10x T4 Ligase Buffer: thawed and resuspended at room temperature
1 parts Vector DNA (mol not l)
3 part Insert DNA (mol not l)
20 µl Nuclease free water
1 µl T4 DNA Ligase
Steps:
- Set up the reaction in a microcentrifuge tube on ice. T4 DNA Ligase should be added last. The molar ratio of vector to insert should be 1:3.
- Gently mix the reaction by pipetting up and down and microfuge briefly.
- For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
- For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours. Alternatively a high concentration of T4 Ligase can be used in a 10 minute ligation.
- Heat inactivate at 80°C for 10 minutes
- Chill on ice and transform 1-5 µl of the reaction into 50 µl of competent cells
KCM Transformation:
Description:
Steps:
- Take 50µl chemical competent E. coli from -80 freezer and thaw on ice
- Add (as master mix):
2,5µl DNA
10µl KCM 5x
37,5µl H2O
- Incubate on ice for 30 minutes
- Heat shock at 42°C for 1 minute
- Incubate on ice for 2 minutes
- Add 900 µl of LB or 2x YT Medium
- Incubate on 37°C for 60min
- Centrifuge 5min at 1000g
- Take 900µl of supernatant and throw away
- Resuspend pellet in remaining media
- Plate out on agar with antibiotics (1:1 / 1:10)
Colony PCR:
Description:
Materials and Chemicals:
PCR-tubes
Forward primer
Reverse primer
Masermix (dNTPs, Polymerase, buffer)
Water
Thermocycler
Endvolume: 10 µl
Steps:
- Pick colonies from plates. Solute one colony in about 20 µl of water.
- Give the colonies into 10 µl colony PCR solution with OneTaq Mastermix (which should be diluted to 1x in the end (e.g. you need 5 µl of 2x mastermix for 10 µl)) and primer (between 0,1 and 1 µl)
- Use the Thermocycler with an appropriate PCR program for at least 25 cycles
week number 27
▼2015-07-01 Ribozyme amplification
|
Number |
Ribozyme |
Primer fwd |
Primer rvs |
|
1 |
CFTR 1 T |
DH_35 |
DH_45 |
|
2 |
CFTR 1 A |
||
|
3 |
CFTR 1 C |
||
|
4 |
CFTR 2 T |
DH_36 |
DH_46 |
|
5 |
CFTR 2 A |
DH_37 |
DH_47 |
|
6 |
CFTR 2 C |
DH_38 |
|
|
7 |
CFTR 1 DE T |
DH_39 |
DH_48 |
|
8 |
CFTR 1 DE A |
||
|
9 |
CFTR 1 DE C |
||
|
10 |
CFTR 2 DE T |
DH_40 |
|
|
11 |
CFTR 2 DE A |
||
|
12 |
CFTR 2 DE C |
||
|
13 |
GFP 1 DE |
DH_41 |
|
|
14 |
GFP 2 DE |
DH_42 |
|
|
15 |
GFP 1 |
DH_43 |
DH_49 |
|
16 |
GFP 2 |
DH_44 |
|
|
|
|
|
|
PCR - amplification
|
|
for 10µl [µl] |
for 50µl [µl] |
|
Template |
1 |
5 |
|
Primer fwd |
1 |
5 |
|
Primer rvs |
0.5 |
0.5 |
|
ddH2O |
2.5 |
14.5 |
|
Q5 Polymerase |
5 |
25 |
PCR conditions
|
|
Time [s] |
Temperature [°C] |
|
Initial denaturation |
30 |
98 |
|
Denaturation |
10 |
98 |
|
Annealing |
10 |
66 |
|
Elongation |
15 |
72 |
|
Final extension |
30 |
72 |
|
Hold |
∞ |
4 |
PCR Purification
- QIAGEN Kit
▼2015-07-05 pSB1C3-vector assembly with a new artificial multiple cloning site and pcat promotor
Description:
In this experiment, we wanted to insert a Multiple Cloning Site (MCS) and a pCat promotor into the pSB1C3-vector
Procedures:
Oligo-Annealing:
Materials and chemicals:
5 µl forward oligo
5 µl reverse oligo
95 µl sterile water
Steps:
- Incubate phosphorylated oligos at 95°C for 3 minutes
- Cool the reaction down slowly for 30 minutes to one hour
- Check the concentration on the nanodrop
Ligation:
Materials and chemicals:
2 µl 10x T4 Ligase Buffer: thawed and resuspended at room temperature
1 parts Vector DNA (mol not l)
3 part Insert DNA (mol not l)
1 µl T4 DNA Ligase
To 20µl nuclease free water
Steps:
- Set up the reaction in a microcentrifuge tube on ice. T4 DNA Ligase should be added last. The molar ratio of vector to insert should be 1:3.
- Gently mix the reaction by pipetting up and down and microfuge briefly.
- For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours. Alternatively a high concentration of T4 Ligase can be used in a 10 minute ligation.
- Heat inactivate at 80°C for 10 minutes
- Chill on ice and transform 1-5 µl of the reaction into 50 µl of competent cells.
Notes:
We used several ligation strategies and insert to backbone concentrations. We used a annealing product for the ligation
20 µl test digest
Description:
Steps
- Set up reaction according to protocol:
ddH2O for a final volume of 20 µl
2 µl of 10x Reaction Buffer (e.g. NEB CutSmart)
0.5 µl of selected Enzyme(s)
ca. 1 µl of mini prep DNA (Range 200-1000 ng)
- Incubate at 37°C for 60'
- Load on gel (add loading dye first)
Notes:
We wanted to verify our results by digestion with:
1. An enzyme that is exclusively on pSB1C3
2. An enzyme that is exclusively on our new inserted multiple cloning site
Oligo Phosphorylation:
Description:
Materials and Chemicals:
2 µl 100 µM oligo stock
2 µl 10x T4 DNA ligase buffer
1 µl T4 polynucleotide kinase
15 µl sterile water
Steps:
- Pipette the Chemicals in one micro centrifuge tube
- Incubate the reaction mixture at 37°C for 1 hour
- Heat inactivate the polynucleotide kinases at 65°C for 20 minutes.
KCM Transformation:
Description:
Steps:
- Take 50µl chemical competent E. coli from -80 freezer and thaw on ice
- Add (as master mix):
2,5µl DNA
10µl KCM 5x
37,5µl H2O
- Incubate on ice for 30 minutes
- Heat shock at 42°C for 2 minutes
- Incubate on ice for 2 minutes
- Add 900 µl of LB or 2x YT Medium
- Incubate on 37°C for 60min
- Centrifuge 5min at 1000g
- Take 900µl of supernatant and throw away
- Resuspend pellet in remaining media
- Plate out on agar with antibiotics (1:1 / 1:10)
Results:
None of our ligations worked to assemble the pSB1C3, MCS and pCat. We used a annealing product without considering that our product has no 5'phosphate and can therefore not be ligated by the T4 ligase. Therefore we used the T4 polynucleotide kinase and tried to ligate again, but the ligation did not worked again.
Conclusion:
We ordered new primers with a 5' phosphate, to try the ligation of the PCR-products (MCS and pCat) with the pSB1C3
week number 26
▼2015-06-22 Miniprep and cryostock of culture 10 in colony PCR
Description:
Procedures:
Qiaprep Spin Miniprep Kit:
Description:
Steps:
- Prepare o/n culture
- Perform mini prep according to manufacturers protocol
E.coli glycerol stocks:
Description:
Steps:
- Grow up an overnight culture of strains of interest
- Transfer 500µl into a safe lock reaction tube
- Add 500µl of 40% sterile glycerol solution
- Freeze slowly at -80°C
Results:
Miniprep: 451,5ng/µl
2 stocks were frozen at -80°
▼2015-06-23 Insert of MCS + promotor in cutted pSB1C3
Description:
Procedures:
Ligation:
Description:
Materials and chemicals:
2 µl 10x T4 Ligase Buffer: thawed and resuspended at room temperature
1 parts Vector DNA (mol not l)
3 part Insert DNA (mol not l)
20 µl Nuclease free water
1 µl T4 DNA Ligase
Steps:
Set up the reaction in a microcentrifuge tube on ice. T4 DNA Ligase should be added last. The molar ratio of vector to insert should be 1:3.
Gently mix the reaction by pipetting up and down and microfuge briefly.
For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours. Alternatively a high concentration of T4 Ligase can be used in a 10 minute ligation.
Heat inactivate at 80°C for 10 minutes
Chill on ice and transform 1-5 µl of the reaction into 50 µl of competent cells.
Notes:
Used fragments/chemicals:
1 µl cutted pSB1C3
0,1 µl MCS
0,1 µl pCat
2 µl T4 Buffer
1 µl T4 Ligase
Filled up to 20 µl with water
Incubated for 2 hours at room temperature
Results:
Plated transformated bacteria (with ligation product) did not grew on a LB-Agar with CM
▼2015-06-23 PCR: Site-change of ribozyme constructs
Description:
Aim: Get every fragment to the same cloning standart.
BamHI -----------------------------/ BmtI
Procedures:
23.06.2015:
Description
PCR mix:
Polymerase Mastermix 2x: 25 µl
Primer: fwd: 1 µl
rev 1 µl
Template-DNA 1 µl
ddH20 22 µl
|
Number |
Fragment Name |
T Anneal |
Fwd |
Rev |
|
1 |
CFTR 1 T |
58°C |
DH13 |
DH14 |
|
2 |
CFTR 1 A |
58°C |
DH13 |
DH14 |
|
3 |
CFTR 1 C |
58°C |
DH13 |
DH14 |
|
4 |
CFTR 2 A |
61°C |
DH13 |
DH15 |
|
5 |
CFTR 2 C |
61°C |
DH13 |
DH15 |
|
6 |
GFP 1 |
61°C |
DH16 |
DH17 |
Results:
1: 1 band + smear: PCR settings were not right --> many sideproducts were synthesized
2: 2 bands + smear: As above, lower band is our desired product
3: 2 bands: Higher product yield than 1 and 2, higher purity than 1 and 2
4: One small band: Low yield and higher purity than 3
5: One small band: Lower yield than 4
6: Smear: No visible product band
The PCR settings were wrong for our target DNA.
Positive colonies exhibit a clear band with a length of about 200 bp. Bigger products seem to be side products
24.06.2015:
Description:
PCR-Mix:
25 µl Polymerase Master Mix 2x
1 µl Primer fwd
1 µl Primer rev
1 µl Template-DNA
22 µl ddH2O
|
Number |
Template |
T Anneal |
Fwd |
Rev |
C(ng/µl) |
Comment |
|
1 |
CFTR 1 T |
60°C |
DH13 |
DH14 |
24 |
Number 1 + 1a in one tube |
|
2 |
CFTR 1 A |
60°C |
DH13 |
DH14 |
30,5 |
Number 2 + 2a in one tube |
|
3 |
CFTR 1 C |
60°C |
DH13 |
DH14 |
25 |
Number 3 + 3a in one tube |
|
4 |
CFTR 2 A |
60°C |
DH13 |
DH15 |
8,1 |
|
|
5 |
CFTR 2 C |
60°C |
DH13 |
DH15 |
8,2 |
|
|
6 |
GFP 1 |
60°C |
DH16 |
Dh17 |
-0,6 |
|
|
1a |
CFTR 1 T |
57°C |
DH13 |
DH14 |
24 |
|
|
2a |
CFTR 1 A |
57°C |
DH13 |
DH14 |
30,5 |
|
|
3a |
CFTR 1 C |
57°C |
DH13 |
DH14 |
25 |
|
|
4a |
CFTR 2 A |
57°C |
DH13 |
DH15 |
7,2 |
|
|
5a |
CFTR 2 C |
57°C |
DH13 |
DH15 |
|
|
|
6a |
GFP 1 |
57°C |
DH16 |
DH17 |
|
|
Results:
1/2 = 1/1a
3/4 = 2/2a
5/6 = 3/3a
7/8 = 4/4a
9/10 = 5/5a
10/11 = 6/6a
1/2: 60°C is the optimal temperature for our PCR
3/4: 60°C and 57°C yield equal amounts of DNA
5/6: 60°C and 57°C yield equal amounts of DNA
7/8: 60°C yields no DNA, 57°C yields less DNA than 1/2
9/10: 60°C yields no DNA, 57°C yields less DNA than 7/8
11/12: Smear, no band at 60°C and 57°C
The Annealing Temperatures for most products are help to get a high yield. Just for 9/10 and 11/12 the yields are not optimal.
Positive colonies exhibit a clear band with a length of about 200 bp. Bigger products seem to be side products.
25.06.2015:
Description:
PCR-Mix:
Like in the former protocols
|
Number |
Template |
T Anneal |
Fwd |
Rev |
|
5.1 |
CFTR 2 C |
55°C |
DH13 |
DH15 |
|
5.2 |
CFTR 2 C |
53°C |
DH13 |
DH15 |
|
6.1 |
GFP 1 |
55°C |
DH16 |
DH17 |
|
6.2 |
GFP 1 |
53°C |
DH16 |
DH17 |
▼2015-06-23 Digest of pSB1C3 BBa_J04450
Description:
Procedure:
20 µl test-digest:
Description
Steps:
- Set up reaction according to protocol:
ddH2O for a final volume of 20 µl
2 µl of 10x Reaction Buffer (e.g. NEB CutSmart)
0.5 µl of selected Enzyme(s)
ca. 1 µl of mini prep DNA (Range 200-1000 ng)
- Incubate at 37°C for 60'
- Load on gel (add loading dye first)
Notes:
Selected Enzymes:
EcoRI/SpeI
Buffer:
Cutsmart
Heat inactivation at 60°C
Everything was given on the gel
A fragment with about 2 kb was cut out
Gel elution: Result: 50 ng/µl DNA
Results:
50 ng/µl DNA
5:
Title: Yeast Transformation with p413-GPD and CFTR construct 2
Author: Hendrik
Date: 23.06.2015
Description:
Procedures:
Yeast transformation:
Description:
10 µl of cells for transformation with a plasmid, 50 µl of cells for transformation with a PCR product
2 µl of plasmid DNA per 10 µl of cells
6 equivalents of PEG
1/9 equivalents of DMSO
100 - 200 µl of liquid medium
Steps:
- Give the plasmid DNA into a Eppi. Add the competent cells.
- Mix, then add the PEG.
- Incubate for 30 mins at room temperature while mixing
- Add the DMSO
- Place the yeast in a 42°C water bath for 5-20 minutes
- Centrifuge cells for 2-3 minutes at 2000 rpm
- Discard the supernatant and resuspend the yeast in the liqiud YPD medium
Notes:
10 µl of Yeast and 100 µl of SD-His medium was taken.
2 transformations were made
After transformation the yeast was plated.
Results:
Some yeast grew on the first plate, but not on the second plate (I took some of the original biofilm and made a fractionated plating). Also the biofilm on the first plate exhibited no further growth. Maybe the wrong yeast medium was token (The writing on the flasks didn't survive the autoclave). Another plating was been made to see if this is right. The yeast on the SD-His and SD-Leu plate exhibited no growth. The yeast in the SD-His/Leu plate grew spotlike and maybe forms colonies.
▼2015-06-23 Yeast Transformation with p413-GPD and CFTR construct 2
Description:
Procedures:
Yeast transformation:
Description:
10 µl of cells for transformation with a plasmid, 50 µl of cells for transformation with a PCR product
2 µl of plasmid DNA per 10 µl of cells
6 equivalents of PEG
1/9 equivalents of DMSO
100 - 200 µl of liquid medium
Steps:
- Give the plasmid DNA into a Eppi. Add the competent cells.
- Mix, then add the PEG.
- Incubate for 30 mins at room temperature while mixing
- Add the DMSO
- Place the yeast in a 42°C water bath for 5-20 minutes
- Centrifuge cells for 2-3 minutes at 2000 rpm
- Discard the supernatant and resuspend the yeast in the liqiud YPD medium
Notes:
10 µl of Yeast and 100 µl of SD-His medium was taken.
2 transformations were made
After transformation the yeast was plated.
Results:
Some yeast grew on the first plate, but not on the second plate (I took some of the original biofilm and made a fractionated plating). Also the biofilm on the first plate exhibited no further growth. Maybe the wrong yeast medium was token (The writing on the flasks didn't survive the autoclave). Another plating was been made to see if this is right. The yeast on the SD-His and SD-Leu plate exhibited no growth. The yeast in the SD-His/Leu plate grew spotlike and maybe forms colonies.
▼2015-06-27 Test if transformed cells have the MCS + pcat insert
Description:
Procedures were done on 5 ml overnight E.coli culture with estimated MCS+pCat insert in pSB1C3
Five colonies were picked from the original plate and were given in 5 snapcaps with 5 ml LB medium each.
The 5 cultures are named 1-5
Procedures:
QIAprep Spin Miniprep Kit:
Description:
Steps:
- Prepare o/n culture
- Perform mini prep according to manufacturers protocol
Notes:
After the miniprep 2 ml of overnight culture were transferred to 100 ml of fresh LB medium with 1:1000 Chloramphenicol
Results:
Nanodrop results:
1: c = 9,5 ng/µl
2: c = 7,7 ng/µl
3: c = 81,5 ng/µl
4: c = 48,5 ng/µl
5: c = 64 ng/µl
A test digestion will be made with 3-5. The concentrations for 1-2 are too low.
20 µl test-digest:
Description
Steps:
- Set up reaction according to protocol:
ddH2O for a final volume of 20 µl
2 µl of 10x Reaction Buffer (e.g. NEB CutSmart)
0.5 µl of selected Enzyme(s)
ca. 1 µl of mini prep DNA (Range 200-1000 ng)
- Incubate at 37°C for 60'
- Load on gel (add loading dye first)
Notes:
Digest with EcoRI and SpeI
Heat inactivation at 65°C for 20 minutes
The gel picture should have one band when the plasmid have no insert (the natural plasmid has no SpeI cutting site) and two bands when the insert is in the plasmid (the MCS has one cutting site for SpeI).
Results:
The agarose gel shows, that none of our colonies has got the MCS + pCat fragment.
The target fragment exhibits 2 bands, the non-cut fragment exhibits just one band. The reason for the larger bands (2 and 4) is unknown.
▼2015-06-27 Transformation test for pSB1C3 + MCS and pcat
Description:
Procedures were done on 5 ml overnight E.coli culture with estimated MCS+pCat insert in pSB1C3
Five colonies were picked from the original plate and were given in 5 snapcaps with 5 ml LB medium each.
The 5 cultures are named 1-5
Procedures:
QIAprep Spin Miniprep Kit:
Description:
Steps:
- Prepare o/n culture
- Perform mini prep according to manufacturers protocol
Notes:
After the miniprep 2 ml of overnight culture were transferred to 100 ml of fresh LB medium with 1:1000 Chloramphenicol
Results:
Nanodrop results:
1: c = 9,5 ng/µl
2: c = 7,7 ng/µl
3: c = 81,5 ng/µl
4: c = 48,5 ng/µl
5: c = 64 ng/µl
A test digestion will be made with 3-5. The concentrations for 1-2 are too low.
20 µl test-digest:
Description
Steps:
- Set up reaction according to protocol:
ddH2O for a final volume of 20 µl
2 µl of 10x Reaction Buffer (e.g. NEB CutSmart)
0.5 µl of selected Enzyme(s)
ca. 1 µl of mini prep DNA (Range 200-1000 ng)
- Incubate at 37°C for 60'
- Load on gel (add loading dye first)
Notes:
Digest with EcoRI and SpeI
Heat inactivation at 65°C for 20 minutes
The gel picture should have one band when the plasmid have no insert (the natural plasmid has no SpeI cutting site) and two bands when the insert is in the plasmid (the MCS has one cutting site for SpeI).
Results:
The agarose gel shows, that none of our colonies has got the MCS + pCat fragment.
week number 25
▼2015-06-19 Quantitative Gel of the PCR of the Gibson assembly fragments
Description:
We performed a gel quantification of DNA yielded in the PCR of our Gibson fragments.
Procedures:
Quantitative DNA gel:
Description:
Steps:
- Prepare an evenly stained agarose gel.
- Load a defined amount of the sample
- Load the DNA Ladder according to manufacturers protocol (e.g. NEB 2-log: 10µl for ng values according to sheet)
- Document and analyze the gel
Notes:
DNA samples were prepared by 1 hour of digestion with Dpn1 at 60 °C, followed by purification using a qiagen kit.
Samples were mixed with 3 parts of 6x loading dye, and loaded together with 2 log ladder.
Samples were: 1/2: PCR product of mCherry
3/4: CFTR testing construct
5/6: GFP
7/8: p413 backbone
Samples were yielded from PCR with Phusion polymerase.
Results summary:
|
Lane |
Lane 1 |
Lane 2 |
Lane 3 |
Lane 4 |
Lane 5 |
Lane 6 |
|
DNA |
2-log |
mCherry |
CFTR |
GFP |
P413 |
2-log |
|
Basepairs |
|
~800 |
200-300 |
~800 |
~6000 |
|
|
DNA Yield |
|
~60 ng/µl |
~60 ng/µl |
~60 ng/µl |
~60 ng/µl |
|
Conclusion:
The bright bands yielded in this gel, in comparison to the previous quantitative gel of the PCR with OneTaq are indicative of a higher suitability of Phusion polymerase for constructs of this composition. Especially backbone p413 yielded a bright band under UV light in our present experiment, whereas in the previous experiment, there were no bands at all. This indicates, that we should choose Phusion for the amplification of larger fragments.
In the following quantification, all the bands yielded a concentration of about 60 ng/µl, fairly sufficient for a following Gibson Assembly.
▼2015-06-21 Colony PCR of E.coli transformated with Gibson-Plasmids
Procedures:
PCR:
Description:
Materials and Chemicals:
PCR-tubes
Forward primer
Reverse primer
Mastermix (dNTPs, Polymerase, buffer)
Water
Thermocycler
Endvolume: 10 µl
Steps:
- Pick colonies from plates. Solute one colony in about 20 µl of water.
- Give concentrated mastermix (which should be diluted to 1x in the end (e.g. you need 5 µl of 2x mastermix for 10 µl)), primer (concentration between and µl). Fill the volume to 10 µl with the water with E.coli
- Use the Thermocycler with the usual PCR program for at least 25 cycles.
- If the result is positive, take the remaining water to seed LB-medium with our E.coli
Notes:
Plasmids: p413-GPD
Insert: mCherry-CFTR-Tags-GFP
Chemicals(one tube):
0,4 µl forward Primer
0,4 µl reverse Primer
5 µl Mastermix
4,2 µl water with E.coli
Results:
Culture 10 seems to be the only picked culture wich exhibits an insert
Positive Colonies exhibit an insert with the CFTR-testconstruct that is about 1,5 kb long.