User:Cassandra M Barrett/Notebook/Haynes Lab/2015/08/24

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GoTaq PCR for T2A-EGFP and mtCas Fragments

Purpose: To amplify fragments via PCR for two side projects. T2A-EGFP fragment is being amplified for Dr. Haynes. Other two fragments are being amplified for Rene's mtCas project from Cold Spring Harbor.

Methods (T2A-EGFP): Amplifying fragment from pX330g reattempt, using GoTaq this time

Set up reaction with P231 and P232

GoTaq MM 25uL Template (undiluted) 1uL FP 2.5uL RP 2.5uL Water 19uL

Run on GoTaq PCR protocol (60C, 30sec)

Results: Band on gel around 850bp as desired, product cleaned up with Qiagen PCR clean up kit

Methods (mtCas): Amplifying two different fragments for mtCas experiment

Using P259/260 and P261/262

GoTaq MM 25uL Template 1uL FP 2.5uL RP 2.5uL Water 19uL

Run on GoTaq PCR protocl (60C, 90sec)

Results: Band on gel for fragment amplified with P259/260 primer set but not for other one. First fragment cleaned up using Qiagen PCR clean up kit and eluted in 30uL of Elution Solution

Concentrations are as follows:

T2A-EGFP fragment (P231/232 Amplified): 248.6ng/uL mtCas fragment (P259/260 Amplified): 245.2ng/uL