User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/07

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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

 * Create a new empty vector that has an intein, chitin binding domain, and his-tag.

Bench work

 * 1)  QuikChange
 * 2) * template = 10 μg/mL pTXB1
 * 3) * forward primer = 12.5 ng/μL ins-His-after-CBD
 * 4) * reverse primer = 12.5 ng/μL Rins-His-after-CBD
 * 5) * anneal step = 7 min
 * 6) * 18 melt/anneal/extend cycles
 * 7) * Kathryn Muratore 12:07, 9 June 2011 (EDT): I had the wrong volume of 10X buffer in the original protocol. Therefore, these reactions were run with 2x buffer, not 1x buffer.
 * 8) analytical minigel
 * 9) * 1.2% agarose gel
 * 10) * Samples loaded by Daniel Catt
 * 11) 10 μL  1KB ladder
 * 12) 5 μL pTXB1 QuikChange experimental reaction
 * 13) * Lost some sample while loading
 * 14) 5 μL pTXB1 QuikChange control reaction
 * 15) -10 --
 * 16) *&rarr; 90V ~1h
 * 17) * stain in EtBr and rinse in TAE
 * 18) DpnI digestion
 * 19) * add 1 μL DpnI to experimental and control tubes from step 1
 * 20) *&rarr; 1h @ 37°C
 * 21) *&rarr; store at 4°C
 * 1) *&rarr; 1h @ 37°C
 * 2) *&rarr; store at 4°C

Results

 * Expect ~6.7 kb band in lane 3
 * Hard to tell here, but there may be a very faint band at the right size in lane 3.
 * Nothing was loaded in the last lane, but we confirmed that the "band" and "smear" are in/on the gel. This can not currently be explained.
 * CONCLUSION: QuikChange failed. Will have to repeat.


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