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

AU CHEM-570 Lab Prep

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Objective Create a new empty vector that has an intein, chitin binding domain, and his-tag. Bench work QuikChange template = 10 μg/mL pTXB1 forward primer = 12.5 ng/μL ins-His-after-CBD reverse primer = 12.5 ng/μL Rins-His-after-CBD anneal step = 7 min 18 melt/anneal/extend cycles 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. analytical minigel 1.2% agarose gel Samples loaded by Daniel Catt 10 μL 1KB ladder -- 5 μL pTXB1 QuikChange experimental reaction Lost some sample while loading -- 5 μL pTXB1 QuikChange control reaction -10 -- → 90V ~1h stain in EtBr and rinse in TAE DpnI digestion add 1 μL DpnI to experimental and control tubes from step 1 → 1h @ 37°C → 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.