Team:GO Paris-Saclay/labnotebook-cpg2

Cpg2 experiments

05/07, by Kenn and Julie (M or R)

Digestion of pGEM-T Easy by HincII

10 µL	pGEM-T-Easy DNA		? ng
1 µL	HincII		? units
2 µL	Buffer Tango 10X
7 µL	Water
Final volume		20 µL

DNA was digested 2h at 37°C.

Electrophoresis gel :

Ligation of pGEM-T Easy / cpg2 (pPS18-08)

2 µL of vector pGEM-T Easy digested (2 ng) were ligate with 4 µL of insert cpg2 digested (? ng). Ligation were let overnight in the fidge.

06/07, by Yueying

Transformation of the ligation product (pPS18-08)

DH5α competent cells were transformed with 5 µL of our ligation product (06/07). After heat choc, 450 µL LB was added and cells were left to grow 1h at 37°C. Cell cultures were spread on two LB IPTG X-gal Ampicillin plates (1/10 of culture then the rest). No negative control made.

08/07, by Céline

Transformation results and cultures

There are plenty of clones for the ligations pGEM-T Easy / cpg2, but most of them are blue (which means closed vector). However, 5 white clones were put in 4 mL LB + Ampicillin, X-gal and IPTG overnight at 37°C and named pPS18-08.1, …, pPS18-08.5.

09/07, by Kenn, William

Extraction of cultures

4 mL of cultures were extracted and put into 50 µL of AE with the extraction kit (Nucleospin plasmid) protocol.

Results :

Sample	Concentration	[protein] / [DNA]
pPS18-08.1	177 ng/µL	1.87
pPS18-08.2	60 ng/µL	1.83
pPS18-08.3	35 ng/µL	1.86
pPS18-08.4	155 ng/µL	1.88
pPS18-08.5	Missing	Missing

pPS18-08.1 and pPS18-08.2 were sent to sequencing (col18-052W)

Verification digestion

4 µL	DNA pPS18-08 (5 clones)
0,5 µL	EcoRI Fast Digest
1 µL	Green Buffer Fast Digest 10X
4,5 µL	Water
Final volume	10 µL

DNA was digested 1h at 37°C.

Electrophoresis gel of digestion :

Migration step lasted 30 minutes at 100V with a 0,8% agarose gel.

on drive

Expected size

Obtained size

pPS18-08

4,5 kpb

pPS18-08.1

pPS18-08.2

pPS18-08.3

pPS18-08.4

pPS18-08.5

4,5 kpb

? kpb

4,5 kpb

Conclusion : We keep pPS18-08.1, .2, .4 and .5.

11/07, by Kenn, William

Sequencing analysis COL18-052W

pPS18-08.1 : Extensive matching. Prefix was partially destroyed : EcoRI site is not garbled. NotI is preserved, XbaI is preserved. Suffix was partially destroyed. SpeI is preserved. NotI is preserved. PstI is garbled. There is a missing C in the terminator, which is probably fine.

pPS18-08.2 : Extensive matching. More analysis needed to verify sequence. Prefix was partially destroyed : EcoRI site is not garbled. NotI is preserved, XbaI is preserved. Suffix was partially destroyed. SpeI is preserved. NotI is preserved. PstI is garbled. There is a missing C in the terminator, which is probably fine. However, there is a missing G in the CDS (pos 494), which is very bad (frameshift of the last third of the protein.)

Conclusion : Glycerol stocks of pPS18-08.1 and .2 are made. pPS18-08.1 will be used for further construction

12/07, by William

Digestions

Preparative digestion, in order to run a XbaI-SpeI orientation-non-preserving ligation later, was done using the aforementioned enzymes.

25 µL	pSB1C3 DNA		600 ng
1 µL	XbaI
1 µL	SpeI
3 µL	CutSmart Buffer 10X
Final volume		30 µL

7 µL	pPS18-08.1 DNA (09/07)		1239 ng
1 µL	XbaI
1 µL	SpeI
1 µL	CutSmart Buffer 10X
Final volume		10 µL

Both DNA were digested 1h at 37°C.

Electrophoresis gel of digestion and gel clean-up :

Migration step lasted 30 minutes at 100V with a 0,8% agarose gel.

Spin column gel cleanup was done according to the protocol [NucleoSpin Macherey-Nagel].

Expected size

Obtained size

pSB1C3

? kpb

pSB1C3

> 10 kpb

2 kpb

1,2 kpb

pPS18-08

? kpb

pPS18-08.1

3 kpb

1,7 kpb

13/07, by Kenn and William

Results :

Sample	Concentration	[protein] / [DNA]
pSB1C3 dig tube 1	10.832 ng/µL	1.63
pSB1C3 dig tube 2	12.201 ng/µL	1.56
cpg2 (from pPS18-08.1)	19.612 ng/µL	1.67

Ligation of pSB1C3 / cpg2 (from pPS18-08.1) (pPS18-13)

4 µL of vector pSB1C3 digested (40 ng) were ligate with 4 µL of insert cpg2 (from pPS18-08.1) (80 ng). Ligation were let overweek-end in the fidge.

16/07, by William

Transformation of the ligation product (pPS18-13)

DH5α competent cells were transformed with 3 µL of our ligation product (13/07). After heat choc, LB was added and cells were left to grow 2h at 37°C. Cell cultures were spread on two LB + Cm (30 ng/µL) plates (1/10 of culture then the rest). No negative control made.

17/07, by William

Transformation results and cultures

There are many clones on plates. 12 clones were inoculated in 6 mL LB + Cm (20 ng/µL)

17/07, by Kenn

Colony PCR

12 clones were tested with this PCR.

Protocol		Programme
DNA	1 µL each if several tubes	Cycle step	Protocol		Cycles
2.5 mM dNTPs	2 µL		Temperature	Time
Primer A (VF2) 10 µM	1,25 µL	Initial Denaturation	95°C	3 min	1
Primer B (VR) 10 µM	1,25 µL	Denaturation	95°C	30s	30
Taq polymerase	0,1 µL	Annealing	49°C	30s
10X GreenTaq buffer	2,5 µL	Extension	72°C	45s
Water	17,4 µL	Final Extension	72°C	5min	1
Final volume	25 µL		16°C	hold

18/07, by William and Kenn

Electrophoresis gel

Migration step lasted 30 minutes at 100V with a 0,8% agarose gel.

Expected size			Obtained size
pPS18-13	1,7 kpb	pPS18-13.1		300 pb
		pPS18-13.2		300 pb
		pPS18-13.3		Ø
		pPS18-13.4		300 pb
		pPS18-13.5		300 pb 1,7 kpb
		pPS18-13.6		Ø
		pPS18-13.7		300 pb
		pPS18-13.8		300 pb
		pPS18-13.9		300 pb
		pPS18-13.10		300 pb
		pPS18-13.11		300 pb
		pPS18-13.12		300 pb

Conclusion : Only pPS18-13.5 show the right signal at 1,7 kpb but it also present a signal at 300 pb which correspond to the vector empty. Thus, we decided to do another PCR but still keep pPS18-13.5.

Colony PCR

14 clones were tested with this PCR.

Protocol		Programme
DNA	1 µL each if several tubes	Cycle step	Protocol		Cycles
2.5 mM dNTPs	2 µL		Temperature	Time
Primer A (VF2) 10 µM	1,25 µL	Initial Denaturation	95°C	3 min	1
Primer B (VR) 10 µM	1,25 µL	Denaturation	95°C	30s	30
Taq polymerase	0,1 µL	Annealing	49°C	30s
10X GreenTaq buffer	2,5 µL	Extension	72°C	45s
Water	17,4 µL	Final Extension	72°C	5min	1
Final volume	25 µL		16°C	hold

Electrophoresis gel

Migration step lasted 30 minutes at 100V with a 0,8% agarose gel.

Expected size			Obtained size
pPS18-13	1,7 kpb	pPS18-13.13		300 pb
		pPS18-13.14		300 pb
		pPS18-13.15		300 pb
		pPS18-13.16		300 pb
		pPS18-13.17		300 pb
		pPS18-13.18		300 pb
		pPS18-13.19		300 pb
		pPS18-13.20		300 pb
		pPS18-13.21		300 pb
		pPS18-13.22		300 pb 1,7 kpb
		pPS18-13.23		300 pb
		pPS18-13.24		300 pb
		pPS18-13.25		300 pb
		pPS18-13.26		300 pb

Conclusion : This time, we see the same as previously for the clone pPS18-13.22.

Cultures

Compared to this colony PCR results, pPS18-13.5 and pPS18-13.22 were inoculated in 4 mL LB + Cm (20 ng/µL) and put overnight at 37°C at 185 rpm.

19/07, by William

Extraction of cultures

4 mL of cultures were extracted and put into 50 µL of AE with the extraction kit (Nucleospin plasmid) protocol.

Results :

Sample	Concentration	[protein] / [DNA]
pPS18-13.5	6.7 ng/µL	1.8
pPS18-13.22	10.8 ng/µL	1.5

These concentration are low so other cultures were inoculated in 5 mL LB + Cm (20 ng/µL).

24/07, by Kenn and William

Extraction of cultures

5 mL of cultures were extracted and put into 50 µL of AE with the extraction kit (Nucleospin plasmid) protocol.

Results :

Sample	Concentration	[protein] / [DNA]
pPS18-13.5	77,4 ng/µL	1.8
pPS18-13.22	174 ng/µL	1.5

31/07, by Britany

Sequencing

pPS18-13.5 and pPS18-13.22 were send to sequence (COL18-05LB)

Digestion to see orientation of the insert

5 µL	pPS18-13.5 DNA (24/07)		387 ng
1 µL	BspE1
2 µL	NEB Buffer 10X
12 µL	Water
Final volume		20 µL

5 µL	pPS18-13.22 DNA (24/07)		870 ng
1 µL	BspE1
2 µL	NEB Buffer 10X
12 µL	Water
Final volume		20 µL

Both DNA were digested 1h at 37°C.

Electrophoresis gel of digestion :

Migration step lasted 30 minutes at 100V with a 0,8% agarose gel.

Gel

Expected size

Obtained size

pPS18-13 good orientation

1,8 kpb

1,6 kpb

pPS18-13.5

? kpb

pPS18-13 bad orientation

2,7 kpb

700 pb

pPS18-13.22

? kpb

Conclusion : pPS18-13.5 seem to be correctly oriented contrary to pPS18-13.22.

03/08, by Mahnaz and Britany

Sequencing analysis COL18-05LB

pPS18-13.5 and pPS18-13.22 both have the insert cpg2. We aslo saw, like previously digestion showed, that pPS18-13.5’s insert has the good orientationcontrary to pPS18-13.22.

We thought our preparation contains both empty and good vector, but the sequencing data suggest us our preparation is pure.

Conclusion : Glycerol stocks of pPS18-13.5 is made. pPS18-13.5 will be used for further constructions.