Bench work
- Check [DNA] of gel-purified samples from 3 days ago
- 95μL H2O + 5μL each DNA sample (ddI, ddV(His), and ddV)
- Plate remaining 800μL of each transformation from 2 days ago
- → 37°C O/N (Tamra will take out)
- I should have plated this yesterday, but I forgot.
Results
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- There does seem to be some DNA, but the exact [DNA] is unclear.
Conclusions and future plans
Possible problems:
- Ligation worked, but transformation did not.
- Solution - transform the ligation product into User:Matt Hartings commercial competent cells
- Ligation did not work, but gel purification did work
- Solution - try to re-ligate again with the correct [DNA] and using the longer incubation at 16°C
- Gel purification did not work
- Also, note that there is a lot of ethanol in these solutions.
- Solution - check [DNA] by UV (1:10 dilution should give A260=0.05-0.1 according to gel estimates based on mass, but will not be visible based on moles.
- Recall that I did not incubate the melted agarose sample in the column before applying a vacuum.
- Solution - repeat all steps starting with NheI digestion!
- This is the last resort and only if UV data is inconclusive or shows no DNA.
- I should be able to use less restriction enzyme in each step. I don't think this caused any problems, but I only have 10μL of SapI left and have been over-digesting with 10U for <1μg of DNA, so I could definitely cut back and still get complete digestion without ordering more at this time.
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AU CHEM-570 Lab Prep
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