Restriction digest of pGFP(LVA) with HindIII

 

Materials:

Methods:

1. assuming DNA in TE buffer ( because added the gel loading dye already by mistake), set up a digest for pGFP(LVA) cut with HindIII in a 1.5ml eppendorf tube, by first pipetting the following:

   

2. Add restriction nuclease HindIII and mix the contents well by gently tapping the bottom of the tube with finger (avoid bubbles). HindIII cuts pGFP(LVA) at two MCS sites (positions 249 and 1062).
3. optinally: briefly spin down the contents of the tube in centrifuge.
4. incubate for 1.0 - 1.5 hours
5. the result should be two double-stranded DNA parts from the original pGFP(LVA) of 3339 bases: GFP(LVA) insert (base positions: 249-1062, total: 813 bases), rest of plasmid (total: 2526 bases).
6. inactivate the restriction enzyme with 15 min (does this work for HindIII?)
7. Perform an analytical gel on digestion sample according to Protocol in Appendix E.1: add loading dye to the digest mixture, pipet 15 ml into the wells, and turn the power on. Store rest of sample in .

Results: ( 1/27/99)

Performed the digest on the two pGFP(LVA) samples from previous step. Because added gel loading dye by accident, used twice as much restriction nuclease HindIII than needed. Incubated digestion reaction for 1 hour. Performed an analytical gel (in the small cast, using 15 ml for each well). Figure 3.1 shows the results of an analytical gel elecrophoresis on the restriction digest samples pGFP(LVA) cut with HindIII and pBR322 cut with EcoRI. The figure shows that for sample 1, pGFP(LVA) appears to have been digested properly, leaving two segments (presumably, a long one, and the GFP(LVA) insert). There is no sign of DNA for sample 2, leading me to believe that the miniprep went bad. Also, stored restriction digests , instead of . Could this have made a difference?

  
Figure 3.1: (a) Analytical gel electrophoresis (1.5 hours, ) on restriction digest samples. Lanes have: 1 = 1kb ladder, 2 = pBR322 cut with EcoRI sample, 3 = pGFP(LVA) sample cut with HindIII, 4 = pGFP(LVA) sample cut with HindIII, 5 = 1kb ladder. (b) 1 kb ladder, not shown to scale with other figure.