User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/24

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Bench work

 * 1) Check [DNA] of gel-purified samples from  3 days ago
 * 2) 95μL H2O + 5μL each DNA sample (ddI, ddV(His), and ddV)
 * 3) *&rarr; measure A260 and A280
 * 4) Plate remaining 800μL of each transformation from  2 days ago
 * 5) *&rarr; 37°C O/N (Tamra will take out)
 * 6) * I should have plated this yesterday, but I forgot.

Results

 * There does seem to be some DNA, but the exact [DNA] is unclear.

Conclusions and future plans
Possible problems:
 * 1) Ligation worked, but transformation did not.
 * 2) * Solution - transform the ligation product into User:Matt Hartings commercial competent cells
 * 3) Ligation did not work, but gel purification did work
 * 4) * Solution - try to re-ligate again with the correct [DNA] and using the longer incubation at 16°C
 * 5) Gel purification did not work
 * 6) * Also, note that there is a lot of ethanol in these solutions.
 * 7) 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.
 * 8) * Do this today!
 * 9) * Recall that I did not incubate the melted agarose sample in the column before applying a vacuum.
 * 10) Solution - repeat all steps starting with NheI digestion!
 * 11) * This is the last resort and only if UV data is inconclusive or shows no DNA.
 * 12) * 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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