Cloning a PCR product after digestion - (May/03/2006 )

Hello All,

Yes, I have seen another thread pinned with same subject but my question is different.

I am trying to clone 2kb insert into 4.5kb vector. My protocol followed so far is as follows, the problems involved therein are in Italics

1. PCR up the 2kb insert using basic heat-start PCR technique. I create restriction sites at the ends of the PCR product.
2. I run the whole PCR on 0.7% agarose gel and then gel purify using Qiagen gel extraction kit. (I get very little product after gel extraction, approx 20ng/ul)
3. I plan to digest 500ng of insert, if available otherwise max possible in 30ul of digestion mix and I run this digested product on the gel and perform gel extraction. I do NOT check the concentration at this point of time.
4. I set up ligation using equal amounts of vector and insert overnight

Obviously no good results because very less insert goes into ligation reaction, I tried doing 3 times and 5 times of the insert as compared to the vector but no gains.

Now what I am planning to get over low concentration after gel extraction issue is to ethanol precipitate PCR product, set up digestion and then gel purify.
I would like to know what experts think about this.

For ethanol precipitation, I found 2 protocols on the web:

1.
7.5M ammonium acetate and ice-cold absolute ethanol
Method:
a. 50ul of PCR reaction +25ul of ammonium acetate+190ul of ethanol
b. Mix and incubate for 30 mins on ice
c. Spin down at 10000rpm at 4C for 10 mins
d. Decant and discard supernatent
e. Air dry pellet and resuspend in 20ul of water

2.
3M Sodium acetate of pH 4.6, 95%ethanol, 70% ethanol
a. 50ul of PCR product +5ul of 3M Sod. acetate + 100ul of 95% ethanol
b. Vortex the rubes and leave at -20C for an hour to precipitate PCR product
c. Spin for 20 mins at max speed
d. Carefully aspirate off the supernatent.
e. Rinse the pellet with 300ul of 70%ethanol
f. Vortex briefly to dissolve the salts
g. Spin for 10 mins, aspirate off the supernatent
h. Dry the pellet in 30ul of water.

I would like to know which protocol is most used and why??
This would probably remove the salts but what about Taq polymerase and Pfu taq polymerase? Would they interfere with digestion reaction performed on PCR product obtained this way?
Please let me know if you recommend gel extraction of PCR product by this method?

hi
first you can try is to digest your pcr product in the primer extension mix (see the neb web page related to that point).
then a purification by column method is reliable (but only if your pcr is ok)
gel purifying after a digestion is better as you need only one purif/precipitation and so loose less product.

Is it necessary to gel purify your pcr product?

Also: once gel purified you already got rid of taq/Pfu.

Another option: perform multiple PCR's and gel extract them.

@fred_33

Thanks for the link, I will setup one 25ul reaction mix and try this out.
I want to refrain from column purification as our PCR purification kit is more than 2 years old and not too sure if buffers are still OK

@vairus

This is the method followed in our lab but my yields are too little so of course I did with multiple PCRs and eluting in single column. This works well but I feel like these are unnecessary steps (multiple PCR, gel purification and gel extraction) so wanted to avoid these steps to save time as I am planning to clone at least 25 DNA fragments

Anybody to comment about Ethanol precipitation protocol that I posted above??

Many thanks

I would include the 70% Ethanol wash step, and just incubating on ice doesn't seem enough for me, though I would add a bigger amount of ethanol in the second protocol.

The second protocol resembles the one I use (I incubate for 30' @ -80). The extra 70% ethanol wash step is to (as stated) remove excess salts (though I haven't tried ammonium acetate instead of sodium).

Thanks vairus,

I will use the second protocol with 500ul of 70% ethanol rather than 300ul
Wondered if there is any specific storage confition for Sodium acetate or room temperature is OK?

Is it really ok to skip gel purification and extraction steps and just go phenol/chloroform extraction?...I also lose a lot of product after gel extraction...from about a few hundred nanograms/ul down to about only 10-15ng/ul...I'd glad if we could skip this step...

I'd use the sodium acetate protocol, and I'm with Vairus, and chill for 30' at -80. The mixture gels, so you know it is cool. NaAc is stable at room temperature.

I'm a little concerned about the suggestion of Fred to add the restriction enzyme to the PCR mix. This will cut the DNA, but the PCR and dNTP mix will fill in the overhang, and make it impossible to clone into a vector.

I agree about the problems with gel extraction. I'd try to avoid it. Phenol chloroform, although it is an old protocol, works very very well. Otherwise, I'd be aiming for a PCR purification column.

Make sure you are not damaging your DNA with UV exposure during your gel extraction. Use 365 nm UV or a Clare DarkReader.