Difference between revisions of "Template:Heidelberg/notebook/cf/week31"

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

	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

	Volume [µl]
Backbone	10ng -> 2
Insert	4ng
T4-Ligase	1
Buffer	1
ddH2O	to 20µl

 

Conditions

• Duration: 15min
• Temperature:  25°C
• 350 rpm

See also LabGuru protocol

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

Colony PCR

 

	Volume [ng/µl]
Primer fwd	0,5
Primer rvs	0,5
OneTaq Polymerase	5
ddH2O	4

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

 

 

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.

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

Colony PCR

 

Miniprep – QIAGEN Kit 

 

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

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:

 

1. 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)

 

1. 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:

 

1. 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.

 

1. Gently mix the reaction by pipetting up and down and microfuge briefly.

 

1. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.

 

1. 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.

 

1. Heat inactivate at 80°C for 10 minutes

 

1. 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:

 

1. Take 50µl chemical competent E. coli from -80 freezer and thaw on ice

 

1. Add (as master mix):

                2,5µl DNA

                10µl KCM 5x

                37,5µl H2O

 

1. Incubate on ice for 30 minutes

 

1. Heat shock at 42°C for 1 minute

 

1. Incubate on ice for 2 minutes

 

1. Add 900 µl of LB or 2x YT Medium

 

1. Incubate on 37°C for 60min

 

1. Centrifuge 5min at 1000g

 

1. Take 900µl of supernatant and throw away

 

1. Resuspend pellet in remaining media

 

1. 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:

 

1. Pick colonies from plates. Solute one colony in about 20 µl of water.

 

1. 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