I inserted a 3131bp fragment (PCR product) to a vector backbone with 3600bp length by two enzymes ECORI and BSP. After plasmid extraction and linearization, the vector run on the Agarose gel. I must be observed a 6731bp band on gel for linearized plasmid, but there is a 4500 bp band.
Foe sequencing of 3131bp fragment (PCR product) in plasmid, I designed 5 primers.
Sequencing for first primer is positive and for others is failed.
This data and checking of plasmid with restriction enzymes showed that only 900 bp of 3131bp fragment (PCR product) is cloned to vector.
Is it possible occurrence of such event?
Try running a gel of your digested insert. Perhaps there is an internal cleavage site that you did not expect. Which Bsp enzyme are you using?
Cloning artifacts pop up invariably, often due to truncated PCR products, though in rarer cases recombination might be to blame. However, if you screen enough clones, you can probably find a colony with the correct insertion provided that 1) your ligation efficiency is good 2) your insert is not toxic to E. coli and 3) the majority of your insert is full-length.
Yes. Since your insert is relatively long, recombination may occur and caused only part of the insert got cloned. It happened to me once with around 2000bp fragment.
About a possible deletion, yes, sometimes bugs can undergo recombinations or even deletion/insertion of extra sequences into plasmids. It's possible that you could find a smaller insert with a noticeable deletion in this case, this also happened to me a couple of time. This depends on the strain of E.coli.
Anyway, what kind of E.coli strain did you used for this cloning? About a possible suggestion, you could try to change the E.coli strain (with a TOP10 strain for example), extract again the plasmid from a liquid culture doing a prep and try again multiple restriction reactions to create a restriction pattern that could confirm you the presence of the whole fragment. Then you should send the prep to the sequencing service for double check
By the way, to me seems quiet strange what you described in the first part of your question.
The reason for this is unknown. Star activity of the enzyme that you are using could also be a reason as well. However, what really bugs me is the idea of trying to put the piece of insert that is so close to the size of your vector. Which ligation method do you use?
It is well documented and many have observed similar phenomenon of not getting the entire fragments cloned in. As suggested by Paolo, try different E.coli strains. In my hand JM109 has worked best so far. I know TOP10 is a better E.coli, but you never know. The other suggestion would be to try electroporation...the reason being possibly the chemical method may be favoring transformation of smaller sized plasmid and not the bigger one. Try incubating ligation mix for longer time, if performing chemical transformation. Have you gel purified your DNAs (both PCR and plasmid) after restriction digestion? This will also confirm any non-specific cleavage by EcoRI or Bsp. you have not specified which Bsp enzyme you are using for cloning purposes. Some of the Bsp are sensitive to dam/dcm methylation and you have be careful what E.coli you have used to grow you plasmid DNA. Also some of these Bsp are funny enzymes, they cut several nucleotides down the recognition seq. Hope this helps.
Try running a gel of your digested insert. Perhaps there is an internal cleavage site that you did not expect. Which Bsp enzyme are you using?
Cloning artifacts pop up invariably, often due to truncated PCR products, though in rarer cases recombination might be to blame. However, if you screen enough clones, you can probably find a colony with the correct insertion provided that 1) your ligation efficiency is good 2) your insert is not toxic to E. coli and 3) the majority of your insert is full-length.
I completely agree with Daniel M Cohen. At this purpose you can perform colony screening with direct PCR (you just should add a 5-10 minutes 94°C step at the begin for coli disruption). You should use a FWD primer on the vector backbone upstream to the insertion site and REV primer on the very tail of your fragment (the same primer used for amplifying from cDNA o gDNA at the begin). Use a 10 or 12 ul PCR for each clone placing a small amount of the colony into a 0.2 ml tube and apply the PCR mixture directly as a mastermix directly into every tube avoiding inter-contaminations. Finally run up to 3 ul max of the PCR on a 0.8% gel in small wells with a proper ladder. You'll be able to screen more than 100 clones in less than half a day. Select only positive clones with proper size band and use them for making a prep and performing a deep restriction pattern + eventually sequencing. I'd like to suggest you to choose more than one positive clone and use 4-5 of them at least for further quality controls.
Classic cloning is only a question of proper planning and great luckiness.
Don't forget to highlight the colonies you're screening on a masterplate or even on the the starting plate to be able to retrieve the correct positive colony.
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