Hi,
I used the above stated kit to purify restriction digestion (BsiWi, 55oC, single-digest, 4h) products (11.7kb for one reaction, ~3.5kb for the other), and got unexpectedly low yield: ~70 % for the 3.5kb product, but only between 19 and 36 % for the 11.7kb product.
I checked the protocol, and Promega states that 9.416kb DNA fragments can be 95% recovered, and 23.130kb fragments 47%. There is no size in between, so hard to interpolate. I wonder though: I did not do gel purification, but used the restriction digestion directly for column purification, which makes the low yield even more worrisome.
Did anyone else experience this problem, maybe in particular with larger DNA fragments?
Thanks,
Patrick
Make sure to run your BsiWi digest in a PCR machine with a heated top. My personal experience with restriction enzymes that function at 55 degrees is they are suboptimal.
Given the size of your largest product (11.7 kb), I'd avoid kits altogether. Here's a variant of the protocol I'd use for gel purification of anything over 10 kb:
1) Run digestion products on 0.7% LMP agarose gel in 1X TBE
2) Cut out fragment of interest with clean cover slip.
3) Melt gel slice at 65oC 5min.
4) Add 2.5 vol. of 20mM Tris pH8, 1mM EDTA, mix by pipetting, place at 65oC 2min.
5) Extract with equal vol phenol pH 7-8.
6) Spin 10min. at RT
7) Chloroform extract
8) Precipitate with 1/10 vol 3M sodium acetate + 2vol Ethanol
9) Wash with 70% ethanol
10) Resuspend in 10ul water. Check recovery on minigel
You can modify the column purification protocol a bit: warm up elution buffer to 65 C before using it. Try adding 98 % ethanol and isopropanol to the binding buffer. Good luck!
One more thing: adjust the PH of the binding buffer to 6, if none of the above works, sometimes lower pH works better with less abundant DNA species in the solution. Don't be afraid to play with it.
At that size your fragment will bind to the silica quite strongly. Try using TE instead of water for elution, pre-warming it at 80 or even 98*C and incubating it with the resin for at least 5 min.
@Leonid: Why lowering the pH? As far as I know, DNA binding to silica is stronger at acid pH...
Dear Patrick, I'm having the same problem with ~8kb mtDNA fragments. Did you manage to overcome the low yied?
Hi
We are experiencing similar problems with a 2.5kB linear DNA fragment and only get yields of about 38%. Will keep you updated on our troubleshooting.
Regards
G
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