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 C_{0}V_{0}=C_{F}V_{F} where C_{0} is initial concentration, V_{0} is initial volume, C_{F} is final concentration and V_{F} 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
C_{0}= 248.5 pM/mL
Clearing for V_{0} we obtain that he have to mix 10 μL of oligo and 490 of water.
Oligonucleotide 5430 Prffix_REV
C_{0}= 351.77
Clearing for V_{0} 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
H_{2}O
 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
H_{2}O
 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.
