Insert size: 1Kb
Vector : 4.5 Kb
Restriction enzyme sites : NdeI XhoI
Backbone was dephosphorylated with rSAP for 1 hr 37C heat inactivated later,
PCR product was gel eluted after digestion from pGEMT clone which had positive fall out.
260/230 ratio 2-2.2 ( good)
Ligation conditions: 16C overnight and 25C 8hrs, 1:3, 1:5 ratio Vector: Insert.
Transformation in DH5alpha ultracompetent cells efficiency 10^8
Colony PCR was done showed amplification- directly from plate, colonies were picked grown overnight- pellet was taken lysed by heating and then PCR was done to confirm (to prevent false positive results from ligated dna contamination)... either case the PCR results were positive, Non Template control doesnt show any amplification (no conatmination in the buffers or reagents)
Yet after 3hrs of double digestion the fall out is not seen at all on the gel. Changed the enzyme to confirm if the enzyme had an issue ...got similar results.
What could possibly go wrong... Please suggest. Incase any further details are required I would provide them. Thanks
Hi Ankita, it seems that you did everything alright - one control experient which might help you to figure more out whats going on is to perform the colony screening using vector specific primers instead of gene specific primers (i.e. T7 or M13, depending on your vector construct). This should give you a small-sized band (i.e. 100-200bp) from the colonies bearing only the empty vector. This helps you to exclude that your gene specific primers amplify something else than your target sequence...Good Luck!
Ankita:
Have you done single digestions? Digest with NdeI alone and XhoI alone. Sometimes double digestion do not work even though the buffer might be compatible. The other thing to do is to increase the digestion time. The other thing that we have sometimes faced is recombination- though with DH5 alpha the chances are minimal. If you still do not get it, you might want to try JM109 cells. We found it is much better than DH5alpha and DH10B cells.
Rohini
What type of Restriction enzyme was used ? Normal / Fast digest ?
# Fast digest (universal buffer, multiple enzyme compatibility, no methylation problem, no star activity, Ultra short incubation time about 10 to 15 min) will give you best result as compare to normal one. we have faced a lot of problem with normal RE enzyme.
# Check whether both the enzymes are working or not ( single digestion).
# Kindly upload the Geldoc picture if possible.
# Try to select single colony from the agar plate and do colony pcr. There is no need of pre boiling of sample before adding pcr master mix. you can directly add the bacteria into master mix.
# Are you getting good amount of plasmid ?
Hi all. Thanks all for suggestions. I am using Restriction enzymes, which seem to be working fine as the pGEMT clone for the same is showing a clean fallout after 3 hrs of digestion. We are getting a good amount of plasmid approx 300ng/ul. Colony PCR is being done from a single colony. We have also used RE from new stocks and got the similar results. We have tried overnight digestion with Nde! And then 3hrs digested with XhoI and got a similar result. We have not tried other cells, although. Also, we haven't tried the plasmid specific primers. The issue is that the REs are working fine in case of pGEMT clone and Colony PCR is yielding positive results then the subclone should yield a positive fallout.
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