Southern blot analysis of PCR products

Protocol 9.


1. Prepare a 3% agarose gel using 3:1 ratio of NuSieve agarose to agarose (FMC Byproducts, Inc.; Rockland, ME).

2. Mix 20 ml of the RT-PCR reaction product with 10 |xl of TAE electrophoresis buffer. Add 6 nl of 6 x loading dye #3 (30) and heat at 68°C for 10 min, followed by quick cooling on ice for 5 min.

3. Load samples into the wells of the gel and perform electrophoresis at ~30 volts/cm until the bromophenol blue marker has run —2/3 to 3/4 the length of the gel.

4. If using unlabelled DNA markers, briefly stain gel with ethidium and photograph with UV transillumination (add 50 ^l of 10 mg/ml ethidium bromide to —500 ml of TAE, using the old electrophoresis buffer). The expected PCR product for the human is 318 bp; and for the mouse 575 bp.

5. Transfer the size-fractionated DNA to GeneScreen Plus™ nylon membranes (New England Nuclear/DuPont, Inc.) using 0.4 N NaOH as the transfer buffer. Alternatively a solution of 0.1 N NaCI and 0.1 N NaOH can be used to transfer to Zeta-Probe membranes (Biorad, Inc.).

6. If using Gene Screen Plus membranes, no baking or UV cross-linking is required. The filters are merely, rinsed in 2 x SSC for 5-10 mln to neutralize NaOH, before proceeding to pre-hybrldization.

For Zeta-Probe or similar nylon membranes, neutralize the filter for 10 min in 2 x SSC, and UV cross-link (1200 joules).

7. For the mouse bcl-2, we hybridize overnight at 57°C with —10 pmoles of 32P-end-labelled internal oligonucleotide probe in 5 x SSPE (1 x SSPE=0.15 M NaCI/0.01 M NaH2P04, pH 7.0/1 imM EDTA), 0.6 % SDS, 50 M-g/ml heat-denatured salmon sperm DNA. Washes of the filter are then performed in 5 x SSPE and 0.1 % SDS at 57°C for 20 min (twice).

8. For the human BCL-2, the procedure we have used entails hybridization with 10 pmoles of 32P-end-labelled oligonucleotide probe in 5 x SSC containing 0.1% SDS, 5 x Denhardt's (30), and 100 ng/ml salmon sperm overnight at 50°C.

Washes are performed with 5 x SSC and 0.1% SDS twice for 10 min at room temperature, followed by exposure to X-ray film [Kodak XAR film (Eastman-Kodak, Rochester, NY)] overnight at -80°C with intensifying screens.

These two alternative approaches have not been compared side-by-side to determine whether one is superior to the other, but both have reproducibly yielded excellent results In our hands.

Was this article helpful?

0 0

Post a comment