What are the main advantages of colony PCR over plasmid miniprep procedures to confirm the ligation of an insert into a plasmid vector?
Be sure to choose test primers covering both part of the insert and part of the vector. That way you can be sure to identify clones having the insert in the right direction. Include a negative control (agar only) to make sure you do not just amplify cloning products not replicated by the bacterial host. Do not exceed 23 PCR cycles, keep amplicon
Hello,
with colony PCR you can screen more clones in a shorter time frame. It is also cheaper in most cases.
Best regards, Christian
Hi,
I share the same opinion that Mr Scheckhuber. Using PCR, you can:
- analyse many clones at the same time
- it's simple, quick and non-expensive
- you can also check the orientation of the insert (if you will express protein)
- avoid digestion problems.
Best regards
After colony PCR you can do minipreps for a couple of clones that screened positive, so it saves you from having to do a bunch of minipreps for DNA that might not even be positive that you would have to just throw out, so it's really cost effective to do PCR colony screening and then prep the DNA for the positives from the colonies from your master plate.
Its quick, cheap and allows screening larger numbers of clones ... but it is very sensitive so false positives can occur. Scrape the top of a colony with a toothpick. plunge it into a small quantity of water (water then used for the pcr) and then put it into 2ml liquid medium for bacterial culture. That way you already have your miniprep cultures for the pcr-positive clones.
Be sure to choose test primers covering both part of the insert and part of the vector. That way you can be sure to identify clones having the insert in the right direction. Include a negative control (agar only) to make sure you do not just amplify cloning products not replicated by the bacterial host. Do not exceed 23 PCR cycles, keep amplicon
As most have said, it is a very quick first screen but you still need to do mini preps on a subset of the positives as the false positive rate tends to be high.
Colony PCR
1. Allows you to screen large number of colonies
2. Saves time and cost
Just a tiny amount of the colony will do fine.
10 ul of reaction vol always worked out, this is really cheap then.
Sometimes most of the inserts are the wrong way into the vector. When the rate of insertion is low for whatever reason, it might be really frustrating to find the right clone. You don´t want to miniprep 48 samples then. But 48 PCRs are just fine....
Best
As everyone mentioned above, the advantages of this technique is that is cheap, quick, and allow you to screen many colonies at once.
Colony PCR is quicker and saves a lot of time, you can screen more in a shorter time. But sometimes PCR gives false positives and hence restriction digestion is the better way to confirm an insert. So the rule is, you can confirm the positive colonies of colony PCR by digestion or go for sequencing.
Hi there,
it is a quicker method, simply pick the colonies and PCR them. I do not favor it myself as i am an advocate of the latter method, i.e. the prep cut and screen as i like the visualisation of the cut fragments on an agarose gel, i find it a neater method, but that is personal of course.
Nikos
Best way to do, first do the colony PCR and grow the positive clones, extract plsmid and do the restriction digestion for insert because some times colony PCR give lot of false positive, after confirmation with restriction digestion send for sequencing..
Gud luck..
Colony PCR might give you positive results even if the cloning makes trouble, for example segregation of positives/negatives bacteria during colony growth if you insert has a disadvantage for the bacteria and it is outgrown. I had this problem when I cloned phage fragments on vector plasmids. Some of these produced lethal effects on the bacteria and were outgrown. But the PCR results indicated the presence (even if 95% of the colony is already negative. If you force the presence of the cloned plasmid by adding antibiotics for its selection you might select clones which carry mutations in your cloned gene in case if the product is somehow toxic for the cell. So if you have no means to look for expression f you cloned gene you must take this into account and check by nucleotide sequencing.
best regards
The only advantage is speed. Cost could be also lower, depending on the method used to check the plasmid minipreps.
- Badges
- Science topic