Restriction Enzyme Digest not working - (Jan/18/2013 )

Hello,

For some reason, my digestions are not working. I am afraid to consult my PI because the cause of the problem might be something that has to do with my procedures rather the actual supplies that I am using. I hope someone can pinpoint what I am doing wrong. Thank you!

Experiment goal:
1. Create insert with sticky ends by introducing restriction sites (Kpnl and Xbal) through PCR, then digesting those ends (with Kpnl and Xbal) after PCR product purification. This should create one band on the gel.
2. Also digest a vector plasmid with the same two enzymes. Since the plasmid is circular this should create two bands, a bigger fragment (which I will extract to use as the vector) and a smaller fragment to toss.
3. Gel extraction, ligation of vector and insert, then transformation.

After running the digestion products of 1 and 2, I saw two bands (instead of one) in the PCR product and only one band in the plasmid lane (instead of two). After making this observation I repeated the entire PCR to start all over, only to visualize the exact same results on the gel even on my second try. This made me want to test the Restriction Enzymes, so here's what I did.

I obtained three different plasmids that each had one Kpnl and one Xbal site, and prepared their digestions in the following order:
1. MB grade water (18.5uL)
2. 10X Thermo Scientific Fast Digest Buffer (3 uL)
3. 2.0 ug vector (= 2.0 uL since the concentrations of all the plasmids were 1ug/uL)
4. Fast Digest Kpnl (2.5 uL)
5. Fast Digest Xbal (2.5 uL)
6. Fast AP Theremosensitive Alkaline Phosphatase* (1.5 uL)
Total volume = 30 uL
Mixed lightly, centrifuged quickly, then incubated at 37C for 1 hour.

*Here I decided to use the Alkaline Phosphatase because I was planning to use the digested fragments for ligation later on if they worked.

However, electrophoresis showed only one fragment for each plasmid lane, all at their original lengths (~4kb).

Because none of them worked, I then decided to repeat the same procedures but now with a new stock of Kpnl and Xbal. I actually prepared three tubes for the same Kpnl and Xbal that I used above, and three other tubes for the new set of enzymes. In total, I ran 6 lanes on the gel. I also altered the incubation duration to 15 min because I realized that I was using reagents for "Fast Digest". However, visualization showed no digestion; I only saw six big bands near the top of the gel, each at their own original fragment size.

Hmm... if it's not the enzymes that aren't working, what could be wrong? I also think the plasmids are in great condition because it's very hard to believe that all three of them aren't in good shape. Plus they have been working when my other lab members used them.

Thank you very much for your time!

~Tom

As you are cloning kpn1 and Xba1 are likely to be very close to each other in the plasmid - the fragment you cut out will be quite small, almost certainly less than 50 bp and as such quite difficult to detect on a gel when you have only started with 2 ug of 4 kbp DNA (you will have 1/80th of the original DNA amount if it is a 50 bp fragment or about 25 ng, which is below the detection limit for an agarose gel).

Check the expected sequence of your PCR product for another Kpn or Xba1 site - there is one there almost certainly. You will need to use a different enzyme for the cloning.

You should not need the alkaline phosphatase in your reaction, since you are using different enzymes. Have you run the PCR product on a gel prior to digestion? Is it a single band? If so, then your PCR product is being internally cut with one of the enzymes, and you will have to use a different enzyme for cloning, as Bob1 says.