I am trying to clone eGFP + a linker into pCite4a. To do this, PCR was performed on a plasmid containing the eGFP with the linker built into the the reverse primer as such:
Forward Primer: 5' NNNN-Not1 site-eGFP 3'
Reverse Primer: 5' eGFP-Linker-NotI Site-NNNN 3'
PCR of the plasmid containing eGFP with these primers produced the correct size product ~760 bp. The PCR positive and negative controls also appear correct. Digestion was done with NEB's NotI HF (I later ran a gel of this digestion and the products are the correct size). Ligation and transformation yielded a significant number of colonies over the vector only control.
Colony PCR was performed to check for direction. I chose a forward primer on the backbone and a reverse primer on the insert. Additionally, I chose a forward primer on the insert and the same reverse primer. This should give two product sizes if the insert is facing the correct direction: 1100 bp and 700 bp (the size of the insert) and only one product size if it is facing the wrong direction: 700 bp.
15 colonies were chosen and PCR products appeared on the gel, however the PCR products appear drastically too small: 250 bp (this doesn't look to be the primers as they can be seen lower on the gel) and 400 bp. The positive control was the undigested backbone with a forward and reverse primer I know work and I choose two negative controls. The first was the undigested backbone with the insert primers to verify that the primers couldn't anneal anywhere else on the backbone and the second was the primers without template to verify that there was no contamination. All of the controls produced the expected results. PCR was then done on miniprep'd DNA and with the same results.
The primers have good melting temps for the initial PCR (60-65) and high temps for the colony PCR (78-80). I've checked for heterodimers, secondary structures and everything else I can think of. I choose NEB's Q5 polymerase for each PCR.
I have double checked all of my work, as well a, having lab mates review it. Does anyone have any ideas why my products would be so small?
Thank you,
Hi Patrick,
Have you tried digesting the clones with Not I to see if the correct size fragment is even there to start with?
You can determine the directionality of the cloning by digesting with an enzyme that cuts in your DNA fragment and one that cuts in the MCS. Depending on the size of the digested fragment you will know which way it is cloned in.
Thanks for the quick response. I forgot to mention that a digestion was done with NEB NotI HF and it produced a short product from 4 minipreps. This doesn't make sense to me when the digested insert is the correct size and the PCR primers are able to bind and produce something, no matter how small.
Thanks
Did you gel-purify the PCR product? You may have a spurious contamination with a smaller product, which will ligate into the vector with higher efficiency than the large fragment. I would always recommend two different restriction sites for cloning, thereby you also prevent cloning of false products with the same primer on both sides.
The backbone was gel purified and the insert was purified with the Promega SV Wizard kit. A gel of the purified products showed no secondary products/contaminants. If it is contamination, then it would occur in either the ligation or transformation steps.
Electroporation was used for transformation, so contamination could have come from the cuvette. But I would think that is unlikely as the transformations were done for 8 different constructs in 8 supposedly clean cuvettes. All constructs show the same small product.
Thank you,
If you have PCR product smaller than positive control try to use DNA Sequencing Analysis and compare to BLAST analysis to appear the missing part
Thanks for all the replies. I went ahead and redid the PCR and then gel-extracted the PCR product. I now have the correct cloned product that has been sequenced.
Thanks again!
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