IGEM:Paris Bettencourt 2012/Notebooks/Semantic group/day by day//2012/08/26: Difference between revisions

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**Mass vector = 37 ng (<=> 1µL)
**Mass vector = 37 ng (<=> 1µL)


it gives : Mass insert = 37 ng => 37/2.8 ~ 25µL
it gives : Mass insert = 37 ng => 37/2.8 ~ 15µL


->2h20 at 22°C
->2h20 at 22°C

Revision as of 08:23, 26 August 2012

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Miniprep of the O/N culture of pSC008

I use the thermoscientific kit, but with the vacuum method. I finally elute in 50μL of water.

  • Nanodrop : 139 ng/μL : -20°C.

Ligation of insert pSC008 into vector pSC010 (pSB1C3)

<html>

<TABLE BORDER="1"> 

 <CAPTION> Ligation compositions, in µL, Final Vol. = 20µL </CAPTION>
  <TR>
<TH> what </TH> 
<TH> C1 </TH> 
<TH> C2 </TH> 
<TH> T- lig </TH> 
<TH> T+ lig </TH>
 </TR> 
  <TR>
<TH> Vector </TH>
<TD> 1 </TD>
<TD> 1 </TD>
<TD> 1 </TD>
<TD> 1(pSC001dig) </TD>
 </TR>
 <TR>
<TH> Insert</TH>
<TD> 15 (*) </TD>
<TD> 15 (*) </TD>
<TD> 0 </TD>
<TD> 10 (L11 dig) </TD>
 </TR>
 <TR>
<TH> Buffer 10X</TH>
<TD> 2 </TD>
<TD> 2 </TD>
<TD> 2 </TD>
<TD> 2 </TD>
 </TR>
 <TR>
<TH> T4 dna ligase</TH>
<TD> 1  </TD>
<TD> 1  </TD>
<TD> 1 </TD>
<TD> 1 </TD>
 </TR>
 <TR>
<TH> Water</TH>
<TD> 1  </TD>
<TD> 1  </TD>
<TD> 16 </TD>
<TD> 6 </TD>
 </TR>


</TABLE>

</html> (*) : calculated thanks to the following formula :

[math]\displaystyle{ Mass_{insert} = ratio \times \frac{Size_{insert}}{Size_{vector}} \times Mass_{vector} }[/math]

with

    • ratio = 5
    • Size insert = 400bp
    • Size vector = 2000bp
    • Mass vector = 37 ng (<=> 1µL)

it gives : Mass insert = 37 ng => 37/2.8 ~ 15µL

->2h20 at 22°C

Transformation of ligation

What's in the tube DNA volume (µL) Cell volume (µL) Plates (LB + Amp)
C1 20 40 180µL pur & 200µL 1/10 & 200µL 1/100
C2 20 40 180µL pur & 200µL 1/10 & 200µL 1/100
positive transformation control (pSB1C3) 0,5 20 200µL
negative Cm transformation control (NEB only) 0 20 200µL
negative ligation control (pSC010 dig. ie. vector) 20 40 200µL
Positive ligation control (pSC001dig+L11dig) 20 40 200µL (Amp)
negative Amp transformation control (NEB only) 0 20 200µL (Amp)


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