User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/07/14

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PCR of standardized plasmid

ABSTRACT

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

  • In order to help my team partner Gustavo in his work and parallelize my work with the pBBRMCS5 plasmid I performed a PCR. The idea is to amplify the backbone of the standardize plasmid without the RFP so Gustavo could introduce a fragment in the standard plasmid. We already have the primers for this purpose, they were synthesized for a previous iGEM team. So the first step was to dilute this plasmids so I performed the following calculations to do this task:


I used a formula that says C0V0=CFVF where C0 is initial concentration, V0 is initial volume, CF is final concentration and VF is final volume. We want a final concentration of 5pM/μL and a final volume of 500μL. So by knowing the initial concentration we can use this formula to find the volume of oligo and water that has to be mixed to obtain the desired concentration.

Oligonucleotide 3768 suffix_FWD

 -C0= 248.5 pM/mL
 -Clearing for V0 we obtain that he have to mix 10 μL of oligo and 490 of water.


Oligonucleotide 5430 Prffix_REV

 -C0= 351.77
 -Clearing for V0 we obtain that we have to mix 7 μL of oligo and 493 of water.


Then I did the PCR. 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 30 cycles.



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