15 replies to this topic

[Amitak47]

Hello Friends, i am new to this forum, i am a beginner in cloning and i am having a lot of problems with it. I am using a PET 22b and PET 28 for cloning my gene. I do all the steps correctly as given in the protocols but at the end i get no insert in the colonies.

I am double digesting (sticky ends) my Plasmids with RE and then also double digesting the PCR product ( 37C for 1:30 minutes) with the same enzymes, i clean both of them using the GEL pure, then i try to ligate them, but after transformation i dont get any inserts when i do a colony PCR. For colony PCR i am using IQ supermix. I have checked if my enzymes are working correctly on the plasmids and it seems they are, but i am not sure if they are digesting my PCR product correctly? Is there a way to find out if the PCR product have been digested correctly? i digest at 37C for 1:30 minutes. Also i have done transformation with a restriction digested plasmid and got no colonies which confirms that the plasmids are properly digested, but after ligation with the insert i get colonies without any insert. I want to tell you that if you think that the plasmids are getting ligated because only one enzyme has worked that is not the case, i use controls where i use ligase without any insert, but i dont get any colonies in them.I dont know what is happening, i am so tired of this. I have been doing this for 2 months now, and today i am starting the whole thing once again from the begining.

Any suggestions, please need help.

[phage434]

First, do you have 5' overhangs on your PCR primers to allow cutting?
Are you cleaning up your pcr reaction prior to cutting?

You can test digestion of PCR products by digesting with each enzyme separately.  Then do a ligation with normal (10x) ligation buffer, not quick ligase.  Heat kill the ligase.  Then run the product on a gel.  You should see both single and double length fragments.  The presence and amount of double length fragments tells you that that specific enzyme is cutting and can be religated.

[Amitak47]

Hello again, thanks for the answer. Yes, i do have overhangs on the primers for the enzyme to cut it. Also i run the PCR product on the gel to check its amplification. After i am sure that it has been amplified i cut it out from the gel and then do a Gel pure. So this way i clean my PCR product also. I have 4 different types of PCR product, i dont have a long overhang in one of them but atleat the rest 3 should ligate with the plasmids properly when double digested with the same enzymes.

But i have not understood your answer correctly, you say run the ligation product on the gel. If i run the ligation product on the Gel i suppose i would see multiple bands on the gel, that is- plasmids that have religated will be circular now and circular plamids appear in multiple forms on the gel and i would also see the plasmids that have not ligated with my insert, so linear plasmids could also be seen. I dont understand what sense it makes to run a ligation product on the GEL?

I have tested the Enzymes under compatiable conditions (Double digestion compatiable conditions) to see if they are cutting well they actually did cut the plasmid well, that is why i was skeptical that may be the PCR product has not been cut, since i couldnt find my insert in the Colony PCR. Could you please eloberate what you said above.

Thanks again.

[phage434]

It is critical to clean the pcr product up BEFORE cutting.  It was not clear from your response if this was being done.

I suggested running the ligation product of the TEST, not of the final ligation on a gel.  The idea of the test ligation is to show that each end of the pcr product (separately) can be ligated.  You ligate the single enzyme cut pcr product with itself, yielding (ideally) double length fragments.

[Papaver]

Some other questions...
1) Do you use one RE for double digest or two different enzymes?
2) Have you tried to digest the plasmids after transformation...I mean in terms of picking some clones, purifing the plasmids and then digest them. If the cloning was correct you should see the insert there.

phage434, on 04 January 2012 - 07:05 PM, said:

It is critical to clean the pcr product up BEFORE cutting.  

Why? I usually clean the product before digestion and it workes.

[phage434]

Yes, then you are doing the right thing.  Although it is possible and sometimes convenient  for gel analysis to simply add restriction enzymes to the PCR reaction after cycling, this does not yield clonable fragments. The pcr enzymes will extend the cut ends of the fragments, blunting the cut site, and making the insert unclonable.

[Amitak47]

Dear Veteran, i think either i am not getting your point or you are not getting mine, sorry for that. After i complete a PCR, i load all the PCR product on the gel run it, cut it from the gel and then do a gel pure. I think a gel pure does clean the PCR product, Please correct me if i am wrong?

Now about checking if the PCR product is getting ligated or not, let me make it very clear, these are my primer

[ CCA TGG AGA TGT ACA CGG] FORWARD   NcoI cutting site
[ CTC GAG TTA CTG CTG CTG CTT ] REVERSE  XhoI cutting site

CCA TGG AGA TGT ACA CGG - NcoI cutting site (forward)

GAA TTC CTG CTG CTG CTT - EcoRI cutting site (reverse)


AAC ATA TGT ACA CGG CGC C - NdeI cutting site

GAA TTC CTG CTG CTG CTT - EcorI cutting site


if i restriction digest them, the cutting site (towrds the left hand side) is a few nucleotides, even if i ligate them back how i will i check that on the gel? Please suggest something?

[phage434]

I'm surprised you are getting pcr product with these primers.  The primer binding region is only 12 bp long in most of these.  In general, this should be 18-20 bp.  Do you  see the expected length product on a gel??

Secondly, the RE cut site is at the far 5' end of each of these primers, with no additional bases 5' of it.  This makes the pcr product almost impossible for enzymes to cut, and in general, they will not be cut.

I would recommend redesigning these primers with 18 bp homology to your site of interest, adding the RE site 5' of that, and adding an arbitrary set of 6 bp or more 5' of the RE site.

Gel cleanup does indeed remove pcr enzymes and buffers.

[Amitak47]

Dear Veteran, thanks for the answer once again. The thing that you are saying about the primers is exactly what i thought in the begining, but when i did a PCR with these primers i did get the expected band, although not a lot. So to get sufficient PCR products i do 3-4 PCR´s and then get the product from that. Also these primers are designed by a post doc student in our lab, so i just cant ask her to get me new once. I did check them with oligocal and with the NEB websites to see if they could be cut, and they could be cut because they dont need a lot of overhangs to get cut, 3 BP is sufficent for the enzymes to cut. Now my problem is that i seem to see do everything correctly and i am repeating the cloning now, but if it doesnt work this time too, i have no idea what to do in that case. That is why i was looking for something that could confirm if the PCR product is getting cut or not!!

[phage434]

You have zero bases of overhang in those primers.  For example, in the first one, CCA TGG AGA TGT ACA CGG, the NcoI site is CCATGG, and has zero bases 5' of it.  The region that is supposed to prime your pcr reaction is AGA TGT ACA CGG, which is 12 bp.  It might be possible (but unlikely) that your template has a well situated NcoI site, so that you prime with all of those bases (18), making the PCR work.  This would explain your partial success with PCR.  But that enzyme almost certainly will not cut the PCR fragment, with zero additional bases.

The NEB web site predicts 0% cutting with zero bases of overhang:
http://www.neb.com/n...nucleotides.asp

[Amitak47]

You are right about the first one, i also know that, i don´t even get a good PCR product in it. I am not worried about the first one, but the others. They are quite ok as per the NEB standards. At least i should get a proper clone in them!!

[Amitak47]

Actually NcoI cutting site is - CCA TGG, so the NcoI will cut after the first C, its like C.....CA TGG. The first C is seperated. So there is a single nucleotidem you can not say that there is no nuclotode! But as per the litrature, minimum 3 nucleoties are should be present, but sometimes even 1 works.

[Amitak47]

I think i will take a proper look at the primers once again.... But thanks a lot!

[phage434]

The count is from the recognition site, not from the cut site.  You have zero. The only primer that has extra bases is the NdeI site, with two.  Even it won't cut very well, 0-20%, and the other end of that pcr product will not cut (EcoRI).

[Amitak47]

Well Veteran, thanks again for the reply. My situation is a little bad in this case, because i tried to explain this situation to the post doc in my laboratory who designed these primers, but she told me that she had designed similar primers before and they had worked and she told me that i should question my own work , so i guess i will continue ,my work and see what happens!