NO Insert found in the Colony PCR
Posted 03 January 2012 - 11:47 AM
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.
Posted 03 January 2012 - 06:13 PM
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.
Posted 04 January 2012 - 06:47 AM
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.
Posted 04 January 2012 - 07:05 PM
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.
Posted 05 January 2012 - 01:16 AM
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.
It is critical to clean the pcr product up BEFORE cutting.
Why? I usually clean the product before digestion and it workes.
Posted 05 January 2012 - 05:23 AM
Posted 06 January 2012 - 11:16 AM
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?
Posted 06 January 2012 - 11:38 AM
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.
Posted 09 January 2012 - 04:50 AM
Posted 09 January 2012 - 06:48 AM
The NEB web site predicts 0% cutting with zero bases of overhang:
Posted 09 January 2012 - 09:31 AM
Posted 09 January 2012 - 09:41 AM
Posted 09 January 2012 - 09:47 AM
Posted 09 January 2012 - 09:52 AM
Posted 11 January 2012 - 07:07 AM