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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] AU CHEM-570 Lab Prep
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Objective

 * Continue trying to insert the His-tag into pTXB1.

Bench work

 * 1) QuikChange of pTXB1 with  His-tag primers
 * 2) One reaction (without a corresponding control) following the  standard QuikChange protocol
 * 3) * compared to last time, the final [buffer] = 1X
 * 4) * 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.
 * 5) * LABEL: QC pTXB1 2
 * QuikChange
 * pTXB1+His
 * 1-step
 * 1) Another reaction (with corresponding control) following the  2-stage modified QuikChange protocol
 * 2) * I accidentally added polymerase to the control tubes (both forward and reverse) at the very beginning
 * 3) These two tubes were removed and stored @ -20°C after the first cycle.
 * 4) * LABELS: ctrl pTXB1 A, ctrl pTXB1 B
 * 1 cycle
 * Pfu added
 * QuikChange neg ctrl
 * pTXB1+His
 * forward, reverse
 * 2-step
 * 1) * I set up new control tubes and put them in the PCR machine for one cycle while the experimental tubes were getting mixed.
 * 2) I subsequently and mistakenly added polymerase to the mixed control tube when it was added back to the PCR machine
 * 3) * LABELS: ctrl pTXB1 A+B
 * 18 cycles
 * Pfu added
 * QuikChange neg ctrl
 * pTXB1+His
 * mixed
 * 2-step
 * 1) meanwhile, I mixed the remainder of the negative control reactions, causing them to miss one cycle
 * 2) * LABELS: ctrl pTXB1 A+B
 * QuikChange neg ctrl
 * pTXB1+His
 * mixed
 * 2-step
 * 1) * experimental reaction was performed according to the protocol
 * 2) ** LABELS: QC pTXB1 A, QC pTXB1 B, QC pTXB1 A+B
 * QuikChange
 * pTXB1+His
 * forward, reverse, mixed
 * 2-step
 * 1) analytical minigel
 * 2) * 1.2% agarose
 * 3) 1KB ladder
 * 4)  Experimental reaction from last time
 * 5)  Control reaction from last time
 * 6) QC pTXB1 2
 * 7) QC pTXB1 A+B
 * 8) ctrl pTXB1 A+B
 * 9) ctrl pTXB1 A+B
 * 10) -10 --
 * 11) *&rarr; 1h @ 90V
 * 12) *&rarr; stain with EtBr
 * 13) DpnI digestion
 * 14) * add 1 μL DpnI to each:
 * 15) QC pTXB1 2
 * 16) QC pTXB1 A+B
 * 17) ctrl pTXB1 A+B
 * 18) ctrl pTXB1 A+B
 * 19) *&rarr; 1h @ 37°C
 * 20)  Transform into  DH10B
 * 21) 200 μL Tamra's fresh DH10B + 5 μL 0.2 mg/mL pTXB1 (from original NEB stock)
 * 22) * to test competency and also make a stock of this plasmid
 * 23) 200 μL Older competent DH10B + 5 μL QC pTXB1 2
 * 24) " + 5 μL QC pTXB1 A+B
 * 25) " + 5 μL ctrl pTXB1 A+B
 * 26) " + 5 μL ctrl pTXB1 A+B
 * 27) * Follow the standard protocol for all
 * 28) * plated on LBAmp
 * 29) *&rarr; O/N @ 37°C starting around 17:43, 9 June 2011 (EDT)
 * 1) * plated on LBAmp
 * 2) *&rarr; 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.


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