Problem with BamHI/SalI double digestion - (Jun/22/2006 )

We are trying to clone PCR fragments which are double digested with BamHI and SalI into a vector double digested by the same enzymes.
Double digestions were performed overnight or during 2 hours at 37°c in BamHI buffer in which we added BSA as recommended by NEB. 5 units per microgrames of DNA were used.
Double digestion of the vector seems to be OK as we can see the digested product on agarose gel. Double digested vector is then purified using Gel extraction kit provided by Qiagen.
For the PCR product, it was amplified using primers in which we added the restrictions sites at the 5' end. We also added additional nucleotides at the 5' end to favorize the digestion giving CGCGGATCC... for BamHI primer and ACGCGTCGAC for the SalI one. The double digested PCR product is then purified using Minelute reaction cleanup kit from Qiagen.
Ligation is performed overnight at 4°c using T4 DNA ligase with 100 ng of the vector and a ratio 3:1 (vector:insert).
After transformation in JM109,
- we did not obtain any clones when transforming the double digested vector (no ligation step)
- we obtained ten clones when transforming the double digested vector (ligation step but with no insert)
- we obtained almost 50 clones when transforming the ligation mixture of the double digested vector and the double digested PCR product.

When analyzing the clones by PCR and sequencing, all correpond to the parental vector as no digestion occurs or as the initial digestion product of the vector religated into the vector.

If anyone has suggestions to help me...it would be great.
Thanks.

Hi spliceman,

did you use a kit to purify your PCR product before the digestion?
Then, what people normaly use is a ratio vector:insert=1:3. I hope you meant this. In theory you should put more insert in teh ligation mixture in order to favor the ligation of the insert in the vector

No, I did not use a kit to purify the PCR product before digestion. The digestion is performed directly onto the PCR reaction.
I used a ratio vectot:insert= 3:1 (ng vector*kb size of insert*3/kb size of vector) as recommended by promega. This ratio was nice for another cloning experiments...

Hi,

with the formula you wrote, you calculate the ng insert as 3 times the ng vactor taking into account the size of the two --> that's a insert=3*vector --> insert:vector=3:1

I neved digested the PCR product without purifying them because you have a salt concentration different from the one the enzyme would like. I wouls try and purify the PCR product first and then do the digestion

OK, I will try this. I'll tell you if it works. I hope so.

Anyone has more suggestions?

according to NEB (the enzyme gods), you can digest with quite a few enzymes in the extension mixture, as long as you supplement them correctly with buffer. check this link for some good info. I would guess your Sal I wasn't cutting so well

also, no matter what formula you use for calculating vector:insert ratio, sometimes you just need to overload it with insert...some ligations are not as efficient as they should be, for whatever reason

this is what I would try: I would cut with Sal I for a short overnight in BamHI buffer. I would add BamHI and go for about another 3 hours the next day. see if that improves your chances? another possiblity is to do sequential digestion (using each enzyme's favorite buffer to increase activity) with a purification in between. you have to start with much more DNA for every purification step you add, but it might help you out

I agree with aimikins. I had a similar problem with salI few weeks back. I did a sequential digest (even though I could have done a double digest) with salI as the second digest. It worked.

good luck !!!

you need more time for digestion for sal1 its worked with me

I tried (as suggested by dnafactory) to purify the PCR product using PCR Qiaquick purification Kit before doing the double digestion...and it works!
Thanks all for your advices.