IGEM:Paris Bettencourt 2012/Notebooks/Semantic group/day by day//2012/09/02: Difference between revisions
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--> 60 min, 37°C water bath. | --> 60 min, 37°C water bath. | ||
Then I cut the bands around 280bp and gel purify them with promega kit. | Then I cut the bands around 280bp and gel purify them with promega kit. I recover in 20µL of water : Nanodrop : 10ng/µL | ||
==Ligation of insert pSC008 into vector pSC010 (pSB1C3)== | |||
<html> | |||
<TABLE BORDER="1"> | |||
<CAPTION> Ligation compositions, in µL, Final Vol. = 10µL </CAPTION> | |||
<TR> | |||
<TH> what </TH> | |||
<TH> L1 </TH> | |||
<TH> L2 </TH> | |||
<TH> L3 </TH> | |||
<TH> Lig - </TH> | |||
<TH> Lig + </TH> | |||
</TR> | |||
<TR> | |||
<TH> Vector </TH> | |||
<TD> 2 </TD> | |||
<TD> 2 </TD> | |||
<TD> 2 </TD> | |||
<TD> 2 </TD> | |||
<TD> 1(pSC001dig) </TD> | |||
</TR> | |||
<TR> | |||
<TH> Insert</TH> | |||
<TD> 3 (*) </TD> | |||
<TD> 3 (*) </TD> | |||
<TD> 3 (*) </TD> | |||
<TD> 0 </TD> | |||
<TD> 7 (L11 dig) </TD> | |||
</TR> | |||
<TR> | |||
<TH> Buffer 10X</TH> | |||
<TD> 1 </TD> | |||
<TD> 1 </TD> | |||
<TD> 1 </TD> | |||
<TD> 1 </TD> | |||
<TD> 1 </TD> | |||
</TR> | |||
<TR> | |||
<TH> T4 dna ligase</TH> | |||
<TD> 1 </TD> | |||
<TD> 1 </TD> | |||
<TD> 1 </TD> | |||
<TD> 1 </TD> | |||
<TD> 1 </TD> | |||
</TR> | |||
<TR> | |||
<TH> Water</TH> | |||
<TD> 3 </TD> | |||
<TD> 3 </TD> | |||
<TD> 3 </TD> | |||
<TD> 6 </TD> | |||
<TD> 0 </TD> | |||
</TR> | |||
</TABLE> | |||
</html> | |||
(*) : calculated thanks to the following formula : | |||
<math>Mass_{insert} = ratio \times \frac{Size_{insert}}{Size_{vector}} \times Mass_{vector}</math> | |||
with | |||
**ratio = 5 | |||
**Size insert = 287bp | |||
**Size vector = 2000bp | |||
**Mass vector = 40 ng (<=> ~2µL) | |||
it gives : Mass insert = 30 ng => 30/10 ~ 3µL. | |||
->2h at 22°C | |||
==Transformation of ligation + Transformation of the QCM PCR & digested plasmid== | |||
{| border = "1" width="45%" | |||
! width="35%" | What's in the tube | |||
! width="5%" | DNA volume (µL) | |||
! width="5%" | Cell volume (µL) | |||
! width="8%"| Plates | |||
|- | |||
|L1 | |||
|10 | |||
|20 | |||
|200µL pur Cm | |||
|- | |||
|L2 | |||
|10 | |||
|20 | |||
|200µL pur Cm | |||
|- | |||
|L3 | |||
|10 | |||
|20 | |||
|200µL pur | |||
|- | |||
|positive transformation control Cm (pSB1C3) | |||
|1 | |||
|20 | |||
|200µL Cm | |||
|- | |||
|negative Cm transformation control (NEB only) | |||
|0 | |||
|20 | |||
|200µL Cm | |||
|- | |||
|negative ligation control (pSC010 dig. ie. vector) | |||
|10 | |||
|20 | |||
|200µL Cm | |||
|- | |||
|Positive ligation control (pSC001dig+L11dig) | |||
|10 | |||
|20 | |||
|200µL (Amp) | |||
|- | |||
|negative Amp transformation control (NEB only) | |||
|0 | |||
|20 | |||
|200µL (Amp) | |||
|- | |||
|positive transformation control Amp (pSC001) | |||
|1 | |||
|20 | |||
|200µL Amp | |||
|- | |||
|#####QCM transformation##### | |||
|##### | |||
|##### | |||
|##### | |||
|- | |||
|QCM-1 | |||
|5 | |||
|20 | |||
|200µL Amp | |||
|- | |||
|QCM-2 | |||
|5 | |||
|20 | |||
|200µL Amp | |||
|- | |||
|QCM-3 | |||
|5 | |||
|20 | |||
|200µL Amp | |||
|- | |||
|QCM-Control | |||
|5 | |||
|20 | |||
|200µL Amp IPTG Xgal | |||
|- | |||
|pUC18 | |||
|1 | |||
|20 | |||
|200µL Amp IPTG Xgal | |||
|- | |||
|} | |||
Revision as of 15:24, 2 September 2012
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Digestion of pSC008
--> 60 min, 37°C water bath. Then I cut the bands around 280bp and gel purify them with promega kit. I recover in 20µL of water : Nanodrop : 10ng/µL
Ligation of insert pSC008 into vector pSC010 (pSB1C3)<html> <TABLE BORDER="1"> <CAPTION> Ligation compositions, in µL, Final Vol. = 10µL </CAPTION> <TR> <TH> what </TH> <TH> L1 </TH> <TH> L2 </TH> <TH> L3 </TH> <TH> Lig - </TH> <TH> Lig + </TH> </TR> <TR> <TH> Vector </TH> <TD> 2 </TD> <TD> 2 </TD> <TD> 2 </TD> <TD> 2 </TD> <TD> 1(pSC001dig) </TD> </TR> <TR> <TH> Insert</TH> <TD> 3 (*) </TD> <TD> 3 (*) </TD> <TD> 3 (*) </TD> <TD> 0 </TD> <TD> 7 (L11 dig) </TD> </TR> <TR> <TH> Buffer 10X</TH> <TD> 1 </TD> <TD> 1 </TD> <TD> 1 </TD> <TD> 1 </TD> <TD> 1 </TD> </TR> <TR> <TH> T4 dna ligase</TH> <TD> 1 </TD> <TD> 1 </TD> <TD> 1 </TD> <TD> 1 </TD> <TD> 1 </TD> </TR> <TR> <TH> Water</TH> <TD> 3 </TD> <TD> 3 </TD> <TD> 3 </TD> <TD> 6 </TD> <TD> 0 </TD> </TR>
</html> (*) : calculated thanks to the following formula : [math]\displaystyle{ Mass_{insert} = ratio \times \frac{Size_{insert}}{Size_{vector}} \times Mass_{vector} }[/math] with
it gives : Mass insert = 30 ng => 30/10 ~ 3µL. ->2h at 22°C Transformation of ligation + Transformation of the QCM PCR & digested plasmid
|