User:Eduardo Vladimir Munoz/Notebook/UNAM Genomics Mexico 2011/2011/08/19

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High stability plasmid development

Abstract

  • B0015 and pSB1T3 backbone ligation
  • Oligonucleotides dilutions
  • repC, and its upstream and downstream regions, PCR

Ligation

Using the electrophoresis gel performed by Fibo we determined the concentration of both the B0015 part and the pSB1T3 backbone both digested with EcoRI and PstI. The B0015 part is ten times as concentrated as the backbone, aproximately. We designed the following reactions varying the volume of the DNA solutions:

Reaction 1 2 3
T4 DNA Ligase 1μL 1μL 1μL
10x Ligase Buffer 2μL 2μL 2μL
B0015 DNA 5μL 4μL 3μL
pSB1T3 1μL 2μL 3μL
mQ H2O 11μL 11μL 11μL
Total 20μL 20μL 20μL


Oligonucleotide Dilutions

The following oligonucleotides were diluted using the concentration formula to 500μL at 5μM: 

C0V0 = C1V1


Number Code Date C0
Extn_Pref_F_Gib 5211 Oct 19, 2011 137.24pM/μL
Extn_Suf_R_Gib 5212 Oct 19, 2011 153.17pM/μL
Upper_RepC 5207 Oct 17, 2011 169.36pM/μL
Low_Repc 5208 Oct 17, 2011 148.08pM/μL


repC PCR

I prepared a 50μL PCR with the Platinum Taq DNA polymerase with the following concentrations:

Reagent Final Conc. Volume
10x PCR buffer 1x 5μL
10mM dNTP mix 0.25mM (each) 5μL
50mM MgCl2 1.5mM 1.5μL
Upper_repC primer 5μM 0.2μM 2μL
Low_repC primer 5μM 0.2μM 2μL
Template repC region unknown 0.5μL
Platinum Taq DNA-pol 2.5 units 0.5μL
Water 33.5μL NA
Total 50μL NA


Two reactions and one control were prepared. The control contains DNA we expect not to be ampliffied by our oligonucleotides.


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