I have been struggling with a cloning and its due to the fact that It is digested only with a single restriction enzyme(I have no other option). I was wondering why do we need 1 pmoles of DNA for Alkaline phosphatase reaction? Can not it be done with 1 ug of DNA(7kb)?
I generally follow NEB protocol which claims that Restriction enzyme and rSAP can be used at same time during digestion. I did some success with less than 1 ug DNA digestion but this time my bad luck was at its peak. All positive clones had wrong orientation(feel the pain).
Would you please share your advice how can I get rid of this problem in addition to my first question?
I used 4C and 16C incubation with 3:1 and 9:1 ligation ratio. Insert size~ 2200bp and vector size~ 5200bp.
I also heat up ligation mixture at 45C for 5 mins before adding buffer and Enz(it was brought back on ice to cool down ,then I added buffer and enzyme)
The reason that NEB lists pmoles of DNA as the measure is that since the phosphatase works on free ends, the relevant measure is the number of ends, not the total mass of DNA. You can imagine how 1ug of a large plasmid has many fewer ends than 1ug of an oligonucleotide. So the relevant measure is how much enzyme and how many ends (ie terminal phosphates) are in the reaction. You can easily convert ug to pm since you know the mass of your plasmid.
Regarding the second problem of all the inserts are wrong orientation. The good news is that the cloning experiment worked. I would suggest just screening a bunch more, preferably from a different ligation or a different transformation to be sure you aren't just isolating siblings from the transformation. It should just be a matter of screening enough.
However if after finding a lot of clones with only the reverse orientation (that you know are independent clones) then you might have a technical issue where the insert is somewhat toxic in the orientation you want. Probably not likely but possible.
The reason that NEB lists pmoles of DNA as the measure is that since the phosphatase works on free ends, the relevant measure is the number of ends, not the total mass of DNA. You can imagine how 1ug of a large plasmid has many fewer ends than 1ug of an oligonucleotide. So the relevant measure is how much enzyme and how many ends (ie terminal phosphates) are in the reaction. You can easily convert ug to pm since you know the mass of your plasmid.
Regarding the second problem of all the inserts are wrong orientation. The good news is that the cloning experiment worked. I would suggest just screening a bunch more, preferably from a different ligation or a different transformation to be sure you aren't just isolating siblings from the transformation. It should just be a matter of screening enough.
However if after finding a lot of clones with only the reverse orientation (that you know are independent clones) then you might have a technical issue where the insert is somewhat toxic in the orientation you want. Probably not likely but possible.
Dear Abir
I'm agree with Michael. The good news is that the cloning experiment worked with one enzyme non correct orientation may happen. you need to screen by pcr many colonies using a primer set that allow to distunguish to you the orientation (eg forward primer annealing on the plasmid and reverse in the insert and vice versa)
you can found an example of colony screening by pcr on my blog: https://proteocool.blogspot.com/?m=1
there you can find also information about PIPE cloning which is an enzyme free cloning approach very simple that will save you from all issues related to the use restrinction enzimes.
good luck
Manuele
Manuele Martinelli thank you very much for your reply. Here is the another problem I have been experiencing . My colony PCR was positive but Restriction digestion did not give me a right size. This is not the first time. It happened multiple times.Even these plasmid with wrong orientation gave positive band in PCR. The band was quite thick and without any other non specific band. Therefore, I did not throw away any of these clones. I just do not know who to trust most. PCR or RE digestion. In my modicum of knowledge, I think RE digestion is the most reliable with min chances of contamination. At least I can relate in silico results with it.
It is hard to state which is more accurate because it depends upon how well you know the sequence (ie is it inferred or did you actually sequence it) and where the primers are located. If the primers are entirely outside the insert, then PCR should give you the accurate size. If one primer is internal, then you are not testing for the region outside of that.
I would suggest trying some other REs and see if different enzymes give you patterns that make sense or not.
Are you including a 'no template' control in your colony PCR runs?
Alejandro Martin , No I did not but i will use no template next time
If you did not then you cannot trust the colony PCR data. What if there is template carryover in any of your PCR reagents? For the time being go with what the RE digests tell you (trying with different enzymes as suggested by Michael J. Benedik is not a bad idea) and do a test colony PCR experiment with a proper control.
Here's to hoping your PCR is not contaminated!
Michael J. Benedik , Thanks for your advice. I used a different restriction enzyme and after screening 22 clones I got one plasmid with the right orientation. 7/22 were positive and 1/7 is the orientation I wanted. Any explanation of this type of nature? If you have an insert with six same codons (CAT in the beginning , can this be a reason behind this wrong orientation?
On a different note, this is a human protein~86kDa and I have successfully expressed and purified in bacteria already.
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