User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/09

AU CHEM-570 Lab Prep

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Objective Continue trying to insert the His-tag into pTXB1. Bench work QuikChange of pTXB1 with His-tag primers One reaction (without a corresponding control) following the standard QuikChange protocol compared to last time, the final [buffer] = 1X This reaction went through and extra first 4 steps (see next step below), was pulled out, wax removed, chilled briefly on ice, and 1μL Pfu Turbo added (followed by more wax) before being returned to the PCR machine for the full standard cycling. Thus, the final amount of Pfu = 5U. LABEL: QC pTXB1 2 QuikChange pTXB1+His 1-step Another reaction (with corresponding control) following the 2-stage modified QuikChange protocol I accidentally added polymerase to the control tubes (both forward and reverse) at the very beginning These two tubes were removed and stored @ -20°C after the first cycle. LABELS: ctrl pTXB1 A, ctrl pTXB1 B 1 cycle Pfu added QuikChange neg ctrl pTXB1+His forward, reverse 2-step I set up new control tubes and put them in the PCR machine for one cycle while the experimental tubes were getting mixed. I subsequently and mistakenly added polymerase to the mixed control tube when it was added back to the PCR machine LABELS: ctrl pTXB1 A+B 18 cycles Pfu added QuikChange neg ctrl pTXB1+His mixed 2-step meanwhile, I mixed the remainder of the negative control reactions, causing them to miss one cycle LABELS: ctrl pTXB1 A+B QuikChange neg ctrl pTXB1+His mixed 2-step experimental reaction was performed according to the protocol LABELS: QC pTXB1 A, QC pTXB1 B, QC pTXB1 A+B QuikChange pTXB1+His forward, reverse, mixed 2-step analytical minigel 1.2% agarose 1KB ladder - Experimental reaction from last time Control reaction from last time - QC pTXB1 2 QC pTXB1 A+B ctrl pTXB1 A+B ctrl pTXB1 A+B -10 -- → 1h @ 90V → stain with EtBr DpnI digestion add 1 μL DpnI to each: QC pTXB1 2 QC pTXB1 A+B ctrl pTXB1 A+B ctrl pTXB1 A+B → 1h @ 37°C Transform into DH10B 200 μL Tamra's fresh DH10B + 5 μL 0.2 mg/mL pTXB1 (from original NEB stock) to test competency and also make a stock of this plasmid 200 μL Older competent DH10B + 5 μL QC pTXB1 2 " + 5 μL QC pTXB1 A+B " + 5 μL ctrl pTXB1 A+B " + 5 μL ctrl pTXB1 A+B Follow the standard protocol for all plated on LBAmp → O/N @ 37°C starting around 17:43, 9 June 2011 (EDT) Results No bands are apparent at expected 6.7 kb. Appears to have not worked, but we'll see what the transformation results are.