Separate digested GFP(LVA) insert with QIAEX agarose gel extraction kit

 

Materials:

(methods partly copied from QIAEX II Agarose Gel Extraction Protocol)

Methods:

1. pour a agarose gel according to protocol in Appendix D.1 [NOTE: It was wrong to use agarose gel here, probably should have used ]
2. get the digested samples, already with loading dye.
3. load the gel wells with 8 ml of 1-kb ladders (in small wells), 450 ml of digested pGFP(LVA) sample 2 (top right), 450 digested pGFP(LVA) sample 1 (mid right), 450 ml of pBR322 digest from experiment 3.2.5 (mid left)
4. run the gel (gel electrophoresis) for 1.5 hours @106V or until dye reached 3/4 of the gel [NOTE: Problem here! GFP(LVA) insert has run off the gel! Next time, run for less time.]
5. separate and excise the GFP(LVA) insert according to protocol in Appendix F.1. Also run pBR322 on the same gel.
6. Store the DNA

Results: ( 1/28/99)

Lost today! The GFP(LVA) insert ran off the gel because I ran it for 1.5 hours, until the dye reached 3/4 of the way. Next time, run it for less time with a 2-comb large cast gel! Figure 3.2 shows the results of running the gel. The pBR322 sample is slightly visible, and the pGFP(LVA) sample 1 large segment is also very visible, but the GFP(LVA) insert ran off. As expected, sample 2 did not show any DNA at all. Because the insert was gone, I did not continue the experiment.

  
Figure 3.2: Seperation gel electrophoresis (1.5 hours, ) on restriction digest samples, with two 2-well combs. Lanes have: top left = nothing, top right = pGFP(LVA) sample cut with HindIII, mid left = pBR322 cut with EcoRI sample, mid right = pGFP(LVA) sample cut with HindIII)