No product after restriction digestion - (Jun/08/2009 )

xyz on Jun 11 2009, 02:32 PM said:

The protocol is pretty much as I wrote above. Make sure the gel is diced into small pieces, as this speeds up the diffusion process into the TE.
The agarose is spun down hard for 10 minutes and the S/N is simply decanted off. Precipitation can be done with either sodium or ammonium acetate. If your yields are expected to be low, co-precipitate with glycogen, using standard procedures.

Thanks

swanny on Jun 11 2009, 10:26 PM said:

xyz on Jun 8 2009, 12:37 PM said:

Make sure the enzyme does not have the star activity .
After you purified from gel, check the concentration,
One more thing is if your digested product is two short, you need a large amount of product if you want to see a band one the gel.

Recently I've got the same problem. After the PCR, I even didn't purify the product, used it directly for the digestion, then performed gel extraction. The amount left of digested product reduced considerably, almost lost from the gel. I have to increase the amount of PCR product 3 times before the digestion and ligation to make sure I have enough DNA.
I tried ligating when I didn't see the band before, sometimes it worked, sometimes it didn't. I agree that the most important one is the ratio. Normally, the molar ratio of 1 vector: 3 insert or 1:5 worked well for me, even when I used just 30ng of vector for 20ul ligation reaction and just transformed 3ul by electroporation.

If you digest a PCR product without purifying the PCR enzymes and dNTPs, then the PCR enzymes will extend the 3' end of digested ends. This will destroy the site, and eliminate any possibility of ligating it with a vector.

phage434 on Jun 22 2009, 10:03 AM said:

I agree that some DNA polymerases can extent 3'dA, but there are some points:
1. To some percentage of PCR products (or digested products), this reaction is succeed (in the case of Taq polymerase, about 30%).
2. After PCR, DNA pol reaches or over its half-life time (I ran 30 cycles and 30 sec at 95 degree per cycle + initiation step at 95 degree for 5-10 minutes), so the active number of enzyme molecules and their activity reduce considerably.
3. The salt condition changes when I add RE buffer.

So, this activities just "destroy" a little amount of PCR products, but I gain the higher amount of DNA if I pass the purification step.
Actually, this works for me many times

swanny on Jun 10 2009, 05:37 PM said:

EtOH precipitation probably can give you cleaner DNA than kits.