Line 689: | Line 689: | ||
<p class="c11"><span class="c12">24/07 – Yueying </span></p> | <p class="c11"><span class="c12">24/07 – Yueying </span></p> | ||
<p class="c2"><span class="c1"></span></p> | <p class="c2"><span class="c1"></span></p> | ||
− | <p class="c11"><span class=" | + | <p class="c11"><span class="c1">There is nothing about this digestion on drive</span></p> |
<p class="c2"><span class="c19"></span></p> | <p class="c2"><span class="c19"></span></p> | ||
<p class="c11"><span class="c39">PCR colony</span></p> | <p class="c11"><span class="c39">PCR colony</span></p> | ||
<p class="c11"><span class="c12">24/07 – Xavier </span></p> | <p class="c11"><span class="c12">24/07 – Xavier </span></p> | ||
<p class="c2"><span class="c19"></span></p> | <p class="c2"><span class="c19"></span></p> | ||
− | <p class="c11"><span class=" | + | <p class="c11"><span class="c1">PCR COLONY ON 10 WHITE COLONIES TRANSFORMED WITH pGEMTeasy FOLC BY XAVIER 23-24/07?? -> missing</span></p> |
<p class="c2"><span class="c19"></span></p> | <p class="c2"><span class="c19"></span></p> | ||
<p class="c2"><span class="c19"></span></p> | <p class="c2"><span class="c19"></span></p> |
Revision as of 15:01, 17 October 2018
folC part
Ligation of gBlock-folC in pGEMT easy
17/07 – Kenn
pGEMT easy
|
2ul
|
T4 Buffer 10X
|
1 ul
|
folC 1.8kb 10 ng/ul
|
1.5 ul 15 ng
|
T4 ligase
|
1 ul
|
Water qsp 10 ul
|
4.5 ul
|
2 h at room temperature |
Streaking clones: gBlock-folC pGEMT easy
18/07 – Kenn
Collection of 5 white clones with insert were streaked on LB Xgal IPTG Amp.
Extraction of the pGEMTeasy- FolC plasmid
20/07 – Yueying (?)
Clone 1, 2 and 4 from 2 ml of culture were used for this extraction with the extraction kit.
Nanodrop results :
Clone 1 |
40,2ng/µl
|
Clone 2 |
19,67ng/µl
|
Clone 4 |
26,43ng/µl
|
digestion of the pGEMTeasy- FolC plasmid
20/07 – Yueying (?)
Plamid was digested by NotI fast digest
|
Clone 1 |
Clone 2 |
Clone 4 |
Plasmid DNA µL |
5 |
7 |
6 |
Fast digest green buffer |
1 |
1 |
1 |
Water µL |
3,5 |
1,5 |
2,5 |
NotI µL |
0,5 |
0,5 |
0,5 |
30 min at 37°C |
digestion pGEMTeasy-folC by NotI results:
expected length : 2 bands at 3000 and 1000 pb
- Clone 1 : not the expected length
- Clone 2 – 4 : not digested
PCR of pGEMTeasy- FolC plasmid
23/07 – Yueying
In order to explain what was obtained with this digestion, we did a PCR with pGEMteasy plasmid as control.
Cycle step |
Protocol |
Cycles |
|
|
Temp. |
Time |
|
Initial denaturation |
95°C |
2min |
1 |
Denaturation |
95°C |
30s |
30 |
Annealing |
44°C |
30s |
|
Extention |
72°C |
2min30 1min/Kb |
|
Final extension |
72°C |
5min |
1 |
Mix below divided in 4 tubes : 1,2 and 4 with DNA extract from clone 1,2 and 4 and tube 4 with plasmid control
Dream Taq polymerase |
0,5 µL |
Taq green buffer |
10 µL |
Water |
69 µL |
M13 rev |
10 µL |
M13F-20 |
10 µL |
dNTP |
0,8 µL |
PCR Results:
Expected size :
- plasmid + insert = 2 Kb
- plasmid = 230 pb
Our control shows a signal stripes at 2,5 Kb which is weird
2 and 4 signal stripes are as expected but quite wick.
Digestion of the PCR product
23/07 – Yueying
We digest the PCR product 2 and 4 with BamHI
|
2 |
4 |
DNA µL |
10 |
10 |
Fast digest green buffer |
2 |
2 |
Water µL |
17 |
17 |
BamHI µL |
1 |
1 |
30 min at 37°C |
digestion PCR product 2 and 4 by BamHI results:
Digestion failed, we can’t conclude anything on clone 2 and 4
pGEMt easy- folC PCR fusion
24/07 – Yueying
Cycle step |
Protocol |
Cycles |
|
|
Temp. |
Time |
|
Initial denaturation |
98°C |
2min |
1 |
Denaturation |
98°C |
30s |
30 |
Annealing |
55°C |
30s |
|
Extention |
72°C |
2min30 1min/Kb |
|
Final extension |
72°C |
10min |
1 |
fusion polymerase |
0,5 µL |
5X buffer HF |
10 µL |
Water |
29,5 µL |
Primer A 10 µM |
2,5 µL |
Primer B 10 µM |
2,5 µL |
dNTP |
4 µL |
DNA |
1 µL |
PCR results :
Expected size :
- plasmid + insert = 2Kb
- plasmid = 230pb
We can see a band at 2Kb, we obtain our plasmid with folC
Gel extraction of pGEMt easy- folC
We used the gel product purification protocol from Nucleospin Gel and PCR clean-up kit
Nanodrop results:
pGEMt-easy-FolC |
12 ng/µL |
Digestion of pGEMt easy- folC
24/07 – Yueying
There is nothing about this digestion on drive
PCR colony
24/07 – Xavier
PCR COLONY ON 10 WHITE COLONIES TRANSFORMED WITH pGEMTeasy FOLC BY XAVIER 23-24/07?? -> missing
Extraction of PCRBlunt-folC
06/08 – Clémence
Clone 2,5,7 and 8 were used for this extraction of PCRBlunt-FolC with miniprep protocol.
folC has been ligated in PCRBLUNT
Nanodrop results
Clone |
Concentration (ng/µL) |
2 |
13,5 |
5 |
13,9 |
7 |
10,4 |
8 |
21,4 |
Digestion of PCRBlunt-folC
06/08 – Clémence
Plasmid was first digested by XbaI and PSTI in order to separated FolC from the plasmid and then digested by SmaI in order to degrade pCR Blunt.
Buffer 2.1 |
4 µL |
XbaI |
1 µL |
PstI |
1 µL |
DNA |
30 µL |
Water |
4 µL |
XbAI and PSTI were inactivated during 20min at 80°C before we added 1µL of SmaI for the third digestion
Gel extraction of folC
06/08 – Clémence
We used the gel product purification protocol from Nucleospin Gel and PCR clean-up kit
Nanodrops results:
Clone |
Concentration (ng/µL) |
2 |
21 |
5 |
17,6 |
7 |
13 |
8 |
13 |
Digestion of PSB1C3 plasmid
06/08 – Clémence
In order to ligate folC in PSB1C3, we first digested this plasmid with XbaI and PstI.
Buffer 2.1 |
4 µL |
XbaI |
1 µL |
PstI |
1 µL |
DNA |
15 µL |
Water |
9 µL |
Size of digest plasmid = 2044pb
Gel extraction of digested PSB1C3
06/08 – Clémence
We used the gel product purification protocol from Nucleospin Gel and PCR clean-up kit
Nanodrops results:
Digested PSB1C3 |
48,4 ng/µL |
ligation of folC in PSB1C3
07/08 – Clémence
For clone 2 and 5 :
Digested PSB1C3 |
1,5 µL |
folC |
6,5µL |
T4 DNA Ligase |
1 µL |
Buffer T4 Ligase |
1 µL |
For clone 7 and 8 :
Digested PSB1C3 |
1 µL |
folC |
7µL |
T4 DNA Ligase |
1 µL |
Buffer T4 Ligase |
1 µL |
Transformation of DH5alpha with ligation mix
08/08 – Britany
We used the heat shock transformation protocol.
100 µL of each transformation were then spread into petri dish.
Few clone were obtain.
PCR colony
08/08 – Britany
Cycle step |
Protocol |
Cycles |
|
|
Temp. |
Time |
|
Initial denaturation |
94°C |
5min |
1 |
Denaturation |
95°C |
30s |
30 |
Annealing |
49°C |
30s |
|
Extention |
72°C |
2min30 1min/Kb |
|
Final extension |
72°C |
10min |
1 |
DNA |
22,4 µL |
Taq |
0,1 µL |
Buffer green Taq |
3 µL |
dNTP |
2 µL |
Oligo 1 VR |
1,25 µL |
Oligo 2 VF2 |
1,25 µL |