Template:Heidelberg/notebook/cf/week33
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 |
|||||||||||||
![](https://static.igem.org/mediawiki/2015/2/27/Heidelberg_150830_HB_colony_pCR_ribozymes_%2B_inserts_in_p413_reihe_1_invert_beschriftet.png)
![](https://static.igem.org/mediawiki/2015/2/2a/Heidelberg_150830_HB_colony_pCR_ribozymes_%2B_inserts_in_p413_reihe_2_invert_beschriftet.png)
![](https://static.igem.org/mediawiki/2015/1/17/Heidelberg_150830_HB_colony_pCR_ribozymes_%2B_inserts_in_p413_reihe_3_invert_beschriftet.png)
![](https://static.igem.org/mediawiki/2015/c/c5/Heidelberg_150830_HB_colony_pCR_ribozymes_%2B_inserts_in_p413_reihe_4_invert_beschriftet.png)
![](https://static.igem.org/mediawiki/2015/2/23/Heidelberg_150830_HB_colony_pCR_ribozymes_%2B_inserts_in_p413_reihe_6_invert_beschriftet.png)
![](https://static.igem.org/mediawiki/2015/6/60/Heidelberg_150830_HB_colony_pCR_ribozymes_%2B_inserts_in_p413_reihe_7_invert_beschriftet.png)
![](https://static.igem.org/mediawiki/2015/0/09/Heidelberg_150830_HB_colony_pCR_ribozymes_%2B_inserts_in_p413_reihe_8_invert_beschriftet.png)
▼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.