Gel Purify Digested pBR322 and GFP(LVA)

 

Materials:

Methods:

1. Pour a agarose gel according to protocol in Appendix D.1
2. get the 6 digested samples from the previous experiment, and pipet 2   of loading dye into pBR322 samples 2 and 3, and pGFP(LVA) samples 1-3. Pipet 5   loading dye into pBR322 sample 1.
3. set up the 123 bp DNA ladder: 2   ladder, 2     loading buffer, 6    .
4. load the gel wells in a 12-well comb, in middle 8 wells, according to the following: for digestion samples each well (2 wells for pBR322), for ladders in well):

   

   pGFP(LVA) #1b and pGFP(LVA) #1c are additional tubes that contain 2   DNA, 6    , 1   NEBuffer 2, 0.5   HindIII, 0.5   XbaI.

5. run the gel (gel electrophoresis) for 1.0-1.5 hrs V or until fast dye has reached 2/3 of the gel
6. separate and excise the GFP(LVA) insert according to protocol in Appendix F.1 (with DNA<400ng):

   

7. store the dna

Results: ( 2/7/99-2/9/99)
By accident, initially used the bad pGFP(LVA) maxiprep attempt, and got no DNA in the gel lanes. So reperformed the experiment on the successful pGFP(LVA) maxiprep, and the gel (Figure ##1578>) showed the expected digested products (lanes: G1, 123 bp ladder, G2, G3). While purifying, the pBR322 products were slightly green. At the end, aliquoted into tubes Tris-HCl (pH 8.5) of solution for 400ng/20   GFP(LVA), pBR322 200ng/20   (2 tubes), all .