User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/07/18

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More PCRs

ABSTRACT

  • Repetition of the amplification of the backbone of the standardize plasmid via PCR using a high fidelity enzyme rTth.

  • I did 3 reactions. I follow this steps:

To make a PCR with rTth enzyme you have to prepare the following reactions:

Mix 1

H2O 9 μL
Buffer rTth[3.3x] 6 μL
Mg(OAc)2[25 mM] 3 μL
dNTPs[10 mM] 3 μL
Oligo suffix_FWD 4 μL
Oligo Prffix_REV 4 μL
Diluted DNA(1μL/20μL) 1 μL
Total 30 μL


Mix 2

H2O 10.5 μL
Buffer rTth 9 μL
rTth DNApol 0.5 μL
Total 20 μL

The next step it tu put Mix 1 to warm at 94°C for 5 min, one second before the first cycle starts, stop the thermocycler and add to the first tube the 20 μL of Mix 2. This is called a Hot start. I left the PCR 35 cycles.

  • I also did two positive controls, the first one consists in use another set of primers which amplify the part that is flanked by prefix and suffix and the same plasmid. The other control consists in use another plasmid but the same set of primers.