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Latest revision as of 17:22, 18 September 2015
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
![](https://static.igem.org/mediawiki/2015/a/aa/Heidelberg_150803_HB_colony_pcrs_de_ribozyme_7.1_7.2_8.1_8.2_8.3_9.1_9.2_9.3_9.4_9.5_10.1_10.2_10.3_10.4_10.5_11.1_12.1_12.2_invert_descr.png)
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
![](https://static.igem.org/mediawiki/2015/d/d9/Heidelberg_150803_HB_DE_ribozyme_cpcrs_1kb_ladder_-_12.3_12.4_13.1_13.2_14.1_14.2_14.3_15.1_15.2_15.3_invert_descr.png)
Positive Colonies: 12-4, 13-1, 14-1, 14-3, 15-2, 15-3