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Please direct all comments, questions, and suggestions to biobzc@hofstra.edu
PLASMID RESCUE PROCEDURE - DIGESTION AND LIGATION
Digestion of chromosomal DNA
Set up a digest as follows:
10 µl chromosomal DNA
3 µl buffer
16 µl nanopure water
1 µl restriction enzyme (ADD THIS LAST)
30 µl total digest
The specific enzyme and buffer that you use will depend on the P-element you are working with. For the common mutant the enzyme is EcoRI. You should have already been told what P-element construct was used in your unknown mutant. Choose an enzyme to use for this line. Check your choice with the instructor before you set up the digest. Pop spin1 the digest and place in the 37oC incubator. These will digest overnight. On the following morning someone from your lab group will need to precipitate the digested DNA. That afternoon someone will need to centrifuge and dry the DNA and then ligate it overnight.
Ligation
Re-suspend your DNA in 178 µl of sterile nH20. Set up your ligation as follows:
178 µl DNA
20 µl ligase buffer
2 µl T4 ligase
200 µl total reaction
Ligate the reaction overnight at 15oC (this will be done in the refrigerated microcentrifuge).
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1 Molecular Biology slang for centrifuge at high RPM for 10 seconds or less.