Hello everyone
I want to insert a 60bp fragment into my vector and we used primers(that includes our insert and 2 RE cut site) after the ligation I want to make sure that my vector contains the insert. So we cut from restriction sites and run on 2% agarose gel for 50 minute in the cold room and use the vector(with no insert) as control. Our expectation is that 760bp fragment for vector with insert and 700bp fragment. In the gel image there is not a clear difference. Does anyone have any suggestion?
Hi there,
60bp difference should be noticable in this range of fragment size on 2% agarose gel. What are actually the RE you use for cloning of the 60bp insert and the RE you use for generating the 700/760bp fragment?
If there is no noticable difference, your cloning has failed. An other way to screen for the right insert is to perform a PCR using insert specific primer and plasmid specific primer.
Hi Damla,
One thing to think about is how many bases are required to flank your restriction digest. If you designed primers with the recognition site right of the 5' end, your amplicon might not digest. My suggestion would be to check an NEB manual to see what's required for flanking bases for your enzymes, or simply redesign the primers to have some extra sequence 5' of your introduces restriction site.
Good luck,
Shale
Instead of doing digestion, why don't you try colony pcr? You just need to use the primer that you have (the one with the restriction site) to do pcr using the colonies as template, after amplification, no band= no amplification=no insert; got band=there is ampliication=the insert is there=> if the insert size is correct, then your cloning is successful
Hi Damla,
As advised by Vikki i will also suggest you that try colony pcr.
1) If high Tm is the case, have you try gradient pcr to see which temperature gives good amplification without non-specific bands?
2) If the above still yield multiple non specific band, then u can try designing another set of primers that binds on vector at location near both ends of the insert so that the fragment that you amplify includes the whole insert and a bit of the vector on both ends
Hi Damla,
I agree with most of the answers posted before, yet I want to go back to the your original question, as it sounds like you may have some basic technical problems....
So here are a few questions for you:
1) What is the ratio of plasmid:insert that you used for the ligation?
2) What are the enzymes you use for the cloning?
3) Is your fragment consisted of two long oligos that you annealed and restricted? If so did you clean them after annealing and restriction (e.g. on a gel or by precipitation). How many extra bases did you design on either side of the restriction site.
4) Did you have a control ligation? If so what was the ratio of colonies in the control ligation vs the ligation with the insert?
Hi, you should be able to assess the presence of your insert by RE digestion and/or PCR. In the first case if in your insert there is a unique restriction site that is not present in the vector you will see a band shift between cutted and uncutted plasmid. Indeed, because of their compact size, supercoiled plasmids may move through a gel much more rapidly than a linear fragment of DNA with the same number of basepairs. If there is no unique RE site, you can choose an enzyme that cut in your insert and in the vector: in this case you will see two bands (if your insert is present) or one band (no insert in the vector).
For PCR, because your insert is very small I suggest to design a 20-bp primer on the insert and a 20-bp primer on the vector and perform PCR on the plasmid with(out) insert: you will see amplification only if the insert is present.
are you amplifying your original insert from template dna?
If so you cannot include the RE cut site and any signal sequence in calculating the Tm as they do not bind to your template in the first round of pcr. The Tm will only be for that part of the primer that can anneal
The idea to cut with RE unique to insert is a great idea (you just need a control with some other plasmid, to make sure the restriction is working). Otherwise I'd recommend to design some primers that would give you 200/260 bp. The bigger the relative difference, the better chance to distinguish empty vector and vector with insert.
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