removing a stop codon - (Jul/06/2007 )

Hello.

I am currently try to clone a gene into a gateway entry vector from a shuttle vector already used in our lab. I have been able to clone the full length gene into pENTR by conventional cloning using an EcoRI-gene-blunt end strategy. I would also like to make version that lacks a stop codon so that I can use gateway to produce a c-terminal tag in case N-terminals cause a problem. I was wondering if any of you had suggestions for reliable ways to remove the stop codon, thats the only change I want to make to the gene.

My current strategy has involved PCRing about 300bp of the c-terminal end of the gene's ORF, removing the stop codon in my primer and adding a NotI site, then digesting this product with an internal cutter (NdeI in this case, cuts about 50bp into PCR product) as well as NotI (sequentially). I also digested my gene out of the shuttle vector with EcoRI and NdeI. After phosphorylating both these fragments and dephosphorylating pENTR (RI-NotI digested) I attempted a 3 part ligation: vector-RI + RI-gene-NdeI + NdeI-cterm-NotI + NotI-vector.

So far I haven't had any luck, all my colonies are either still lacking the c-terminus or just vector alone, which is off since I usually get no colonies from my vector alone ligation. My fragments all appear to be the correct size on a gel prior to ligation. I typically use 3-5x the amount of inserts than vector (by molar ratio). Is there something I'm overlooking, like compatible ends in this strategy?

Anyway, I get the feeling there may be an easier way to generate my gene w/o stop codon, so I was hoping someone would have a suggestions. I've considered PCR amplifying the whole gene, but am a little wary since it is about 2.4-2.8 kb (I'm working with a few different isoforms). Would site directed mutagenesis be better? Any preferred protocols?

Thanks for any and all help!

if you are ligating digested inserts, there is no need to do a phosphorylation as said fragements are already phosphorylated.

I assume you have given enough skirting basepairs to the NotI site on the PCR fragment (containing the C terminal tag). NotI needs a longer then average skirting of bp around the restriction site for the enzyme to cut.

I guess my question would be, have calibrate the mol ratio of fragment (RI-gene-NdeI) to fragment ( NdeI-cterm-NotI)? It is somewhat important to get the fragment to fragment ratios to 1:1 to get a multiway ligation working. Same too for NdeI, but I guess with NdeI being internal (50bp being choped off), that shouldn't be a problem.

Could you tell me your dephosphorylation conditions? It is very easy to over dephosphorylate and damage the over hangs rendering the vector unligatable. The rule of the thumb is as follows : 1pmol gets dephosphorylated by 0.1 Unit CIP in 60min at 37 celsius in a volume of 50ul in NEB buffer3.

I would also avoid using any quick ligase buffers. Multiways don't like it. Stick to an overnight ligation at 16 Celisus using normal ligase buffers.

As for direct PCR, 2.5kb is small. One should not be worry over something that size. I would only be concerned if I had to PCR a 15kb fragment. That takes some working on preparing good template and monkeying around with the amplification conditions. For 3kb, any proof reading polymerase like KODhifi or Vent should get you your product, almost without any need for optimising conditions. The primer should aim for a tm of 58 to 60 Celsius. The tm difference between primers should be no more then 2 Celsius, with strong perference of zero difference. Only the section of the primer that binds to the template is used to calculate the tm.

Site directed mutagenesis can be easy but that depends on the plasmid size. Guessing the size of your plasmid at 5kb, I believe this method too is doable without much problems. Controls is an absolute must for this method. DpnI digest can be less then perfect.

I might actually go with the PCR route. The 2.5kb PCR should be easy. Plus you can modify the ends the way you would like to have.

I havnt worked with NdeI. NEB site says "DNA purified by standard miniprep procedures is cleaved at lower rates". Just take care about this fact.

You dont need to dephosphorylate the vector as its a sticky end ligation.

Thanks for the advice, I think I will design a couple more primers and try the full PCR route. My lab seems to be afraid of doing full-length PCRs because of the possibilities of error, but the more I read the more it seems to be the way people are doing it these days. We use either Vent or Pfu Turbo polymerase for cloning PCR, should these be adequate or should I look into a newer enzyme?

As a rule of thumb I usually add 6 nucleotides after the restriction site, but maybe this wasn't enough for NotI.

I dephosphorylate with NEB's antarctic phosphatase. I typically add 1uL, and increase my reaction volume by 10ul including 1uL of phosphatase buffer (the enzyme is supposed to work in any NEB buffer provided you supplement w/ the enzyme buffer), immediatly following digestion for 15min at 37deg, in this case I then gel purified my DNA (using qiagen kit). The DNA itself looked clean and fairly good yield following purification (assessed by running it on a gel).

My ligations were either room temp for at least 2 hours or ~24 hours at 16deg, I've gotten equivalent results both ways, its more a matter of when in the day I can set up the ligation.

At 6bp the cutting efficiency of NdeI is 0%
At 6bp the cutting efficiency of NotI is 10%

I would agree 6bp is not enough.

It is hard to tell from the information provided if overdephosphorylation has occured. You do need to know how much vector was being dephosphorylated.

As for polymerase, between the two choices I would go for Vent. The Pfu turbo is aimed for longer products. So for a 2.5kb product the yeild would probably be not as good as Vent.

As perneseblue suggested, I would go with vent.

Vent is my preferred enzyme anyway, in my past experience it has given me better amplification than Pfu.

It sounds like the most likely problem is the NotI digest, the Nde digest is internal so I'm not concerned about that. I probably am not getting efficient digest of the PCR product w/ only 6bp, based on the NEB catalogue it looks like I need at least 10 extra for digestion, so I've redesigned my primer accordingly. I think the vector itself may also not be cut well, as I've done RI/NotI double digest to open it and the two sites are fairly close to each other, so its quite possible I've been working with only RI digested vector, which could explain the empty vector from some of my ligations.

Thanks again for all your help!