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