Difference between revisions of "Team:GO Paris-Saclay/labnotebook-cpg2"
| Line 91: | Line 91: | ||
<p class="c11"><span class="c0">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.</span></p> | <p class="c11"><span class="c0">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.</span></p> | ||
<p class="c6"><span class="c0"></span></p> | <p class="c6"><span class="c0"></span></p> | ||
| − | <p class="c11"><span class="c41">09/07, by Kenn, William | + | <p class="c11"><span class="c41">09/07, by Kenn, William </span></p> |
<ul class="c17 lst-kix_list_2-0"> | <ul class="c17 lst-kix_list_2-0"> | ||
<li class="c11 c28"><span class="c0">Extraction of cultures </span></li> | <li class="c11 c28"><span class="c0">Extraction of cultures </span></li> | ||
| Line 263: | Line 263: | ||
<p class="c11"><span class="c3">Conclusion : We keep pPS18-08.1, .2, .4 and .5.</span></p> | <p class="c11"><span class="c3">Conclusion : We keep pPS18-08.1, .2, .4 and .5.</span></p> | ||
<p class="c6"><span class="c3"></span></p> | <p class="c6"><span class="c3"></span></p> | ||
| − | <p class="c11"><span class="c58 c66 c56 c9">11/07, by Kenn, William | + | <p class="c11"><span class="c58 c66 c56 c9">11/07, by Kenn, William </span></p> |
<ul class="c17 lst-kix_list_2-0"> | <ul class="c17 lst-kix_list_2-0"> | ||
<li class="c11 c28"><span class="c3">Sequencing analysis COL18-052W</span></li> | <li class="c11 c28"><span class="c3">Sequencing analysis COL18-052W</span></li> | ||
| Line 271: | Line 271: | ||
<p class="c11"><span class="c0">Conclusion : Glycerol stocks of pPS18-08.1 and .2 are made. pPS18-08.1 will be used for further construction </span></p> | <p class="c11"><span class="c0">Conclusion : Glycerol stocks of pPS18-08.1 and .2 are made. pPS18-08.1 will be used for further construction </span></p> | ||
<p class="c6"><span class="c0"></span></p> | <p class="c6"><span class="c0"></span></p> | ||
| − | <p class="c11"><span class="c41">12/07, by William | + | <p class="c11"><span class="c41">12/07, by William </span></p> |
<ul class="c17 lst-kix_list_2-0"> | <ul class="c17 lst-kix_list_2-0"> | ||
<li class="c11 c28"><span class="c0">Digestions</span></li> | <li class="c11 c28"><span class="c0">Digestions</span></li> | ||
Latest revision as of 15:28, 17 October 2018
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 4,5 kpb ? kpb 4,5 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.