I am doing SDM by stratagene kit.After PCR amplify product was observed on agarose gel.Beside that i run my original plasmid on gel too.Unfortunately after PCR i was not getting correct amplify product.
I have doubt regarding my plasmid.As in picture the correct size 6.5kb band is 2. (from above)but amplification is happenning at 3rd band.which is incorrect size of my gene.
what should i do???Please suggest me.
What is exactly the problem? That you do a plasmid prep and you get three bands?
Yes i did get three band,and i proceed with PCR ,but amplification is happening at lower band(incorrect size,nearly 5kb)
My gene+vector size is 6.5 kb.(second band)
So should i do miniprep again?
Shoyab Ansari, OK, so getting the three bands is not the problem. Is the problem then that you are doing SDM on that plasmid and you expect that the reaction will yield a 6.5 kb product but instead it yields a 5 kb product?
Exactly,,it should be 6.5 kb band amplification,but that is not happenin.(Although very less amplification with respect to 5 kb ).
Shoyab Ansari, I think we are not sure exactly what you are showing on the gel. Is the plasmid shown on the gel digested and linearized with a restriction enzyme? If so and you are seeing both the 5 and 6.5 kb bands then it would seem that your original plasmid preparation is contaminated with vector alone and vector plus insert. You might need to retransform and prepare a new plasmid prep to make a pure stock.
Dear Michael
Third well of agarose gel contain only plasmid.I used this plasmid for SDM , and i did get amplify product after PCR(first two well).
My question is regarding my plasmid stock,bec my gene plus vector size is 6.5 kb,which is second band in lane three.But after Pcr i am getting more amplification in third band.(first two lane is PCR Amplify product)May be this 5kb gene is the part of my gene,degraded or something,???and during Pcr primer r able to bind to this form too??)
Still i will do fresh miniprep from same plasmid glycerol stock.
But i am totally unaware about this third band,what it can be,if it is amplified by pcr?
Sorry not fr running the DNA ladder
I would make a dilution of your plasmid stock and retransform it into fresh cells so that you get transformants with single plasmids. Then miniprep a few of those to identify one that have the correct plasmid. I think it most likely that your current plasmid prep has two different plasmids in it, so you need a stock that you are sure has only the correct one.
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