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

 

Materials:

Methods:

1. Pour a agarose gel according to protocol in Appendix D.1
2. get the 3 digested samples from the previous experiment, and pipet of loading dye (resulting in samples)
3. load the gel wells in a 12-well comb according to the following (for digestion samples each in 2 wells, for ladders in well):

   

4. run the gel (gel electrophoresis) for 1.5 hrs or until fast dye has reached 2/3 of the gel
5. separate and excise the GFP(LVA) insert according to protocol in Appendix F.1.
6. store the dna

  
Figure 3.3: (a) Gel before restaining, showing only ladders and pGFP(LVA) without GFP(LVA) insert. (b) Gel after staining, now also showing pBR322 vector and GFP(LVA) insert. [Note: figs not to scale]

Results: ( 1/29/99)

Ran the gel for 1:20 hours (see Figure 3.3(a)) but because I forgot to add EtBr to the gel (red) chamber, the results don't show the DNA. The pGFP(LVA) for all samples shows as a strong band somewhere between 1kb and 2kb. The GFP(LVA) insert is not showing. The pBR322 for all samples appears as a very faint band, probably between 3kb and 4kb. I pipetted EtBr into TE buffer chamber of the gel apparatus (on red side), but that didn't really help.

I therefore restained the gel (according to protocol in Appendix E.2). Figure 3.3 (b) shows the restained gel. Now, both the pBR322 vector and the GFP(LVA) insert are visible. I excised the pBR322 into 2 tubes and the GFP(LVA) insert into 3 tubes, and added the following according to QIAEX II protocol:

Then, added QIAEX II. Then, eluted with Tris-HCl, pH 8.5, centrifuged, pipetted the supernatant into clean tube, and stored . All DNA tubes have about solution volume. The GFP(LVA) tubes should have 30ng DNA each ( DNA), and the pBR322 tubes should have 50ng DNA each ( DNA).