PCR and Digest cleanup before ligation. - A shortcut by avoiding gel purification. (May/29/2007 )
Hey all you molecular biologists. I'm Alex... new guy on the forums. Here goes my first post.
I am trying to clone importins into a pGEX-4T3 vector and have been having lots of difficulty getting colonies after transforming with my ligation reactions. It really sucks, but such is research. At least I have a plan: clone the importin insert into a more "palatable" vector and then subclone it into the pGEX-4T3 vector. Until I find that other vector, I wanted to repeat the cloning one more time.
My question is: can gel purification be skipped when cloning from PCR? If one uses a PCR/enzymatic reaction cleanup kit (I am thinking of using the one by QIAGEN), then you would not need to absolutely run the vector and insert restriction digests on a gel and then gel purify, correct?
Here are the tentative steps for my cloning:
Insert preparation:
1. Amplify the insert by PCR.
2. Run an analytical gel of PCR reactions (using 5ul of a 50ul reaction)
3. If PCR is succesful, use QIAGEN's PCR reaction cleanup kit to purify the amplified insert.
4. Digest the insert with restriction enzymes (EcoRI and NotI in NEBEcoRI buffer).
5. Use QIAGEN's enzymatic reaction cleanup kit to purify the digested insert.
6. Run the DNA on a Spectrophotometer (100x dilution).
Vector preparation:
6. Digest pGEX with restriction enzymes (EcoRI and NotI in NEBEcoRI buffer) + alkaline phosphotase (CIP)
7. Purify the digested vector with QIAGEN's enzymatic reaction cleanup kit.
8. Run the DNA on a Spectrophotometer (100x dilution).
Ligation:
9. Use 1:1, 1:3, and 1:5 insert:vector ratios.
10. Ligate with T4 Ligase at 4C O/N.
11. Transform 3.3ul of 10ul total ligation reaction into 50ul of DH10a cells and plate on Amp+ plate.
Would anyone with experience in cloning please look over this and offer any insight? Especially about not running the digests of the vector and insert on a gel. Using QIAGEN's enzymatic reaction cleanup kit would save me a lot of time while cloning AND it would improve the yield of DNA (their gel purification kit sucks). Also does the last step look OK? I know there is some debate about how much of a ligation reaction should be used to transform competent cells (I will use 1/3 of the reaction).
Thanks a lot for your help, guys!
Alex
I would heat kill the restriction enzymes, 80C for 20 minutes.
You might not have to use CIP with a double digestion, and I would avoid it if possible. If you need to dephosphorylate, you should use shrimp alkaline phosphatase or antarctic phosphatase. AP requires zinc in the reaction mix.
You can probably avoid purification after RE digests and go directly to ligation if you heat kill the enzymes. In general, the few stages the better.
EcoRI buffer has Triton-X100 as a component, and will give problems in chemical transformation. I'd shift to buffer 3, which is better for NotI anyway, which is a fussier enzyme.
Normally I skip the purification of PCR product if there is single band.
I would heat inactivation the RE digestion and purify it with the PCR purification kit, since my lab doesn't have kits specific for purification of RE products.
Some of my labmates run a portion of the ligation mix by agarose gel electrophoresis to check the successful of ligation reaction. However, they always forget that the DNA concentration is low and they end up with empty lanes - the DNA amount loaded is below the detection level!! 
You might not have to use CIP with a double digestion, and I would avoid it if possible. If you need to dephosphorylate, you should use shrimp alkaline phosphatase or antarctic phosphatase. AP requires zinc in the reaction mix.
You can probably avoid purification after RE digests and go directly to ligation if you heat kill the enzymes. In general, the few stages the better.
EcoRI buffer has Triton-X100 as a component, and will give problems in chemical transformation. I'd shift to buffer 3, which is better for NotI anyway, which is a fussier enzyme.
The only reason I want to use phosphotase in the digestion of the vector is to remove the phosphates from the stretch of DNA that was removed between my two restriction sites so that it will not be reinserted into the vector during ligation... if I purify the digest without phosphotase, I'm afraid it might give me a great deal of false positives after ligation.
BUT, now that you made me think about it, since the restriction enzymes are in the MCS, the stretch of DNA between the restriction sites is much smaller than 100bp, which is the smallest size recoverable by the PCR/enzymatic digestion cleanup kit (100bp-10kbp). So yeah, I think I might skip the phosohtase treatment. Any thoughts?
You really want to clean up the PCR reaction prior to the restriction digest. If you don't, then the remaining PCR enzymes and dNTPs will gladly extend the 3' end of the cut fragments to blunt end you restriction sites, making them difficult or impossible to religate.
I see.. Thanks..
Hi Alex,
Your plan is fine. As others have said, I would skip the purification after digestion and just heat-inactivate the enzymes. Purification steps always cause a loss of yield. Some other tips:
-I don't know what spectro you have, but if it's a Nanodrop I probably wouldn't bother. It won't give you an accurate reading for DNA fragments purified by a silica column (it's okay for minipreps). I stopped quantifying DNA for ligations long ago. When you do your 1:1, 1:3 and 1:5 ligation ratios, have a look at the number of columns - from my experience it won't change much. I've accidentally done a 1:1500 ratio before and it worked fine.
-If your ligase buffer contains polyethylene glycol, you'll get a better result by incubating for 5 minutes on the bench rather than overnight at 4c. I suggest transforming some of the ligation after 5 minutes, then put the rest in the fridge and transform it the next day and compare the number of colonies.
-Regarding the amount of ligation mixture to transform: in theory, less is better. Once though, I transformed 2ul (out of 10ul) and the remaining 8ul, and found that 8ul was much better! Who knows!
-Don't heat shock your cells. They don't like it. Instead, mix your DNA with 50ul of your cells and plate directly on to pre-warmed Amp plates (Pope & Kent, Nucleic Acids Res, 1996). I found it's slightly more efficient and it saves a LOT of time.
Thanks for that, phage434... though do you think the small amount of digestion buffer that makes it in to the ligation buffer is enough to make a difference?
to the competent cells, probably yes. The cells are already fragile and a bit of detergent wouldn't help much.
Hey Zouden, just curious, why does nanodrop not work for the silica-column purified DNA preps? I was thinking to use this technique and just wondered why?
Hey guys, thank you for all of your suggestions! Currently I am trying out the protocol I posted (without the phosphotase treatment)... this time I am using two different vectors that contain the MCS: my vector of interest that contains a GST tag (pgex4t3), and a vector that has been recently used for cloning (pmal) as a positive control.
You can probably avoid purification after RE digests and go directly to ligation if you heat kill the enzymes. In general, the few stages the better.
I will go ahead and heat killl the enzymes next time.... but if I'm doing a double digest on a vector, then wouldn't I have to purify the cut-outs to avoid their re-insertion into the vector during ligation? By cut-outs I mean the stretch of DNA between the two restriction sites in the vector's MCS. Once I didn't purify after digestion and got >500 on my ligation plate... of the 10 colonies I screened, none contained my insert. I think it's because the tiny frgament that was cut out stayed in the reaction and was religated into the vector.
I think Zouden meant "if it's NOT a nanodrop, then I wouldn't bother."