I am trying to Linearize plant expression vector using PmlI and Spe I restriction sites which are very close to each other with only two nucleotides in between like cacgtgTAactagt. I found some issues in this because the restriction digestion produced some non specific bands which is not expected in this step. Please guide me on this issue.
Hi there,
Try to limit the amount of enzymes in order to increase the specificity of site recognition for cutting. You may also try sequential digest (PmlI first and then SpeI) if you feel the enzymes actually compete for cutting.
Hi......
Can you show the pic of restriction digestion? What is the size of restriction fragments you got on gel? Prefect restriction will give you only two bands, if enzymes u used are unique and provided that any of the enzyme you used for digestion should not have isochizomeric sites. Generally select enzyme pair for digestion such that these two sites are at least 25 to 30 bp away from each other for efficient digestion by enzymes. Use the same units of both enzymes for double digestion in single time. While loading on gel use plasmid as uncut, linearized vector using any one of the enzyme and double digested product that will provide you exact idea what is happening with the diestion. Better perform r.d. in double as well as serial digestion and check it on gel. Else try another enzymes for linearizing the vector. Your question will be answered correctly if you show restriction pattern.
U can also use the either side of optimum limit of two enzymes.. i.e. use enzymes at not their optimum condition... Most probably it will work...
Thanks all for the help. I reduced the digestion time, amount of enzyme and performed sequential digestion and it worked well. The reason for multiple band may be over digestion or STAR activity.
Thanks.
Suyash
I think a general answer to this problem for any 2 enzymes is to check the New England Biolabs web site for " how many bases extra are needed to cut pcr product ". Some enzymes cut right up to the end of a sequence and some need 1,2,3 or more bases as well as the cut site . Then cut with the enzyme that needs most extra bases followed by cutting with the enzyme that needs fewest bases
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