Team:Paris Saclay/Notebook/September/3
From 2014.igem.org
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+ | ===Digestion=== | ||
+ | In order to clone LS in pPSI, i Digest it by PacI | ||
====Ligation==== | ====Ligation==== |
Revision as of 15:29, 9 September 2014
Contents |
LabWork
B- Construction of the fusion protein
We made a classic exttraction of plasmids from the liquid cultures.
Check by electrophoresis if it worked and send it for sequencing.
D- Lemon scent
by Mélanie
PCR LS
Electrophoresis of the PCR made Yesterday
well 2-3 = PCR with Dream taq or Vent taq
(well 4-5 = checking of the pps5 Extraction)
The PCR have success so I use the PCR clean up kit to purify it
Electrophoresis after purification
Digestion
In order to clone LS in pPSI, i Digest it by PacI
Ligation
We already have some pPSI digested and dephosphorelated
So I do a ligation:
component | volume |
---|---|
H2O | 5μl |
buffer | 2μl |
ligase | 1μl |
pPSI | 2μl |
LS PCR | 10μl |
2 hours at room temperature and over night at 4°
pPS5
Plasmid exctraction using the kit (picture is here)
digestion by SalI
component | volume |
---|---|
H2O | 11μl |
buffer | 5μl |
SalI | 2μl |
pPS5 | 30μl |
Electrophoresis
We saw that we have something strang with our plasmid but we wil check it tomorrow with a good ladder (the ladder here was normally on the well 1)
pPS3 and pPS4
We digest the plasmid by HindIII to direct our insert
component | volume |
---|---|
H2O | 6μl |
buffer | 1μl |
HindIII | 1μl |
pPS3/4 | 2μl |
well 1 = ladder well 2-3-4 = pPS3 Well 5-6-7 = pPS4
results are very strange