I did a PCR and inserted restriction sites on either side of my gene of interest. I then digested the PCR product and performed ligation into two different plasmids. 5:1, insert vector.
I transformed, got a few colonies , 3 to 4. Control plates were good and clear of colonies.
I did colony PCR using vector primers and saw positive results. I then do restriction digest using BamH1 and Sall and I only see my plasmid (5000+ bp) on the gel but no insert (300+ bp)
Someone, please help. I've been failing at this for weeks now. :(
It could be several different things. First you could have a positive colony PCR due to contamination have you ruled this out? I would use different pipettes and TAQ to see if you still have a positive. Then as far as the restriction digestion are you sure the restriction sites are formed again? Also how are you purifying the DNA are you able to purify 300bp fragments with the method you are using?
It would be better to check for insert with one insert specific primer and one vector specific primer to make sure that the insert is actually in the vector. 3 positives is very low and it may be that the transform has failed
For a small band using a low agarose percent gel at high voltage for a long time will make it very difficult to see the bands at 300bp. Especially if the concentration is low. So you were able to see your plasmid but not your insert. Thus, for the small bands, use 2% or 3% agarose gel and don't run for a very long time.
For colony PCR, did you confirm that the band size was correct? Since you have a small insert (300+ bp), I would recommend that at least one of the primers is insert-specific rather than vector-specific.
One more point, check the efficiency of your digestion. For example, if the restriction enzymes expired or still working efficiently and the digestion buffer gives the optimal activity for both enzymes without star activity.
Alaa Allahham Firstly, thank you for your advice. I am running a 1% agarose gel at 80V for 2hrs (its a 150ml gel). I used vector-specific primers and tested the gene-specific primers as well, it does seem as if my gene is there.
Restriction enzymes were checked as well :( :(
I read that I cIould try a PCR with gene specific primers on my isolated plasmid to see if the gene is there. What do you think?
Colony PCR has a very high false positive rate due to the presence of unincorporated plasmids.
Grow up a liquid culture and do a plasmid prep.
As my graduate advisor said "you can't rush quality"
Good luck!
Alia Mike, I am positive it is the gel and the long-running time. Here is a small example, the attached two photos are for the exact same gel (1% agarose). The first one was taken after running at 130 volts for 20 minutes, while the second after 1 hour. You can see that the band at 1600 bp was clear after 20 minutes and start to vanish with a longer running time, and that was a 1600 bp band, not 300. The lower bands would be even more difficult to see especially if your input was low from the beginning.
Alia Mike, about your question, yes it's fine to do PCR for your purified plasmid. If your goal is to confirm that the obtained colony has the right plasmid with your insert, the common working flow is Colony PCR>> select the positive colony and grow on liquid LB (with appropriate antibiotics) overnight >>> plasmid extraction and purification >>> sequencing >>> based on the sequence results store the colonies that hold the right plasmid in 20% glycerol at -80°C. The sequencing will confirm your insert and ensure there is no undesired mutation.
A few possibilities: 1) you are just unable to see the band on the gel. Note that as the band gets smaller it is much less bright on a gel since there is less total mass in the band. I'm not convinced that at least in the bottom gel you would see the band. You might try staining the gel again after the electrophoresis, as you note you are losing signal that is probably due to loss of EtBr.
2) Another possibility is you have the clone but one of the restriction sites is messed up. You could digest your plasmid with each enzyme alone and see if they both cut or one does not cut. Also have you looked carefully to see if your presumed plasmid + insert runs 300bp larger than the vector alone? You should be able to see that difference.
3) you might have a mixture of plasmid. If most of the plasmid in your prep does not have insert but a small fraction does, then you won't be able to see the band on a gel but it would still provide template for PCR amplification.
Take another set of unique Restriction enzymes, one in the backbone, one in the gene and do a digestion to see. Choose these restriction enzymes a little bit further apart, so you can produce a ex. 700 bp or larger band (easy to see).
1-First of all, make sure about your gel conditions as the size of GOI is small.
2-In the plating step, along with the transformed bacteria, you will also spread the untransformed remaining DNA on the plate. Thus, these untransformed DNA will be as a template in the PCR reaction, obtaining the desired band. I would suggest you, instead of the colony, do PCR by using extracted plasmids.
3-In digestion reaction, try to use positive control, and optimize your condition (incubation time, units of the enzyme, inhibition by PCR components or salt, act.).
Good luck
I would recommend Katie s comments. The main reasons why u don't get restriction positive is because you might have used excess insert than recommend or excess ligation mix that is not incorporated. Make sure you only use 50 ng ligation mix. I would also make primers that span both vector and insert. Isolate plasmids and follow PCR or RE.
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