We received a plasmid delivered by mail and performed transformation experiment following protocol. the plasmid is used is topo with 3.9kbp. we used AK plates for blue/white screening due to the ampicillin and kanamycin resistance. After selection of white plates, innoculation and plasmid extraction was performed. Clones' plasmid were checked for band using pcr electrophoresis. It is consistent that all clones produced bands way larger than the target gene band.
We used gene specific primers
Cloning is much simpler when it comes to mentioned protocols, but most of the times labs that deal with generation of many clones, can accidentally cross contaminate the clones. It is possible that the clone you received is cross contaminated. Since even after amplifying with the gene specific primers the product size is higher than expected, it means that there is something else in the clone. As suggested above, you need to get it sequenced with vector backbone primers to be sure about what exactly is sitting inside MCS.
Good Luck
Hi, I agree with the opinions above to sequence the PCR product(s) and as well perform restriction digestion but the best is to sequence.
As a first step I would simply cut the plasmid with a single-cut restriction enzyme and check its linear size on an agarose gel. Then I would use other restriction sites to check the size of the cloned gene. If the plasmid has dimerized (or multimerized) during long-term growth, then PCR might give more than the expected band size.
Hi,
I'm agree with everyone that checking your vector sequence would help. There is also one quick test that you actually should always include in your cloning protocol that is a control ligation without insert. So basically you make a ligation mixture the same with your complete mix except for the insert. Then you transform this mixture as a control.
- If the colonies number and composition is the same between control and insert transformation then you get a hint that something wrong with your ligation or your vector
- If you got more colonies or white colonies in your insert mix than in your control, then the vector and ligation is fine. The problem would be your PCR that might be taking wrong insert.
Good luck!
I think Elizabeth And Albino are more relevant in their suggestions. From what I understand, you are only transforming a plasmid you received, so you can amplify the plasmid and use it to your cloning requirements downstream. I would definitely get it sequenced if you are getting a size much larger than you expect and also test with a single digest. It is a good practice when you get a plasmid from someone else: even your lab mate, to test it prior to putting it to use.
I agree with idea about negative controls. Also I suggest to check first the length of plasmid itself, because it would make it clear, does plasmid size match expected value, or not. It's important to choose unique restiction site outside insert sequence, to make sure there is only one copy of target gene.
Also, is the elongation time for PCR correct? It's not very likely, but still possible, that polymerase could run strand-displacement amplification. Or, if the plasmid was dimerized and the elongation time is too big, you can see a large product, but the band of right size should appear also.
Sometimes it's really useful to pick up primers for outside the insert, cause some genes may have really hard-to-read sequence, especially ones with short repeats.
thank you all for your comments. Actually whatI did is perform PCR of the plasmid received before transforming to the host.
We were able to confirm the target before transformation but the clones all have way longer bands as compared to the pcr product before transformation. Also with the presence of two antibiotics where the plasmid is resistant to, we also thought that the those growing would most likely containing the plasmid.
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