Hiii everyone, i have cloned 917 bp gene in pTZ57R/T (2886bp) which is TA cloning vector. If i digest my plasmid with ECOR1, which is vector cutter at 615 bp position and Sac1 which is gene (insert) single cutter at 640 bp. On digestion what size of fragments i should get?
hi,
since you have not provided restriction digestion sites profile in your insert, I have a few questions. Do you have EcoR1 and Sac I site in the the fragment that you have cloned? site selection for restriction digestion for screening clones is largely based on unique sites preesent in vector as well as insert. in the link atached I see Sac I site in the vector also. i would suggest you to linearize your plasmif with EcoR1 alone and look for the size of the band. in case your fragment has been cloned in the vector you would get a band of size 2886 + 971 ~ 4kb. that could be a confirmation for your clone
secondly you have to repeat the digestion with an enzyme that has site only in the insert and get a 4kb product again. that would be the confirmation of your clone.
http://www.snapgene.com/resources/plasmid_files/ta_and_gc_cloning_vectors/pTZ57R_T/
Dear Sansriy,
Thanks a lot for your answer. In my insert there is no EcoR1 site. its only sac1 at 640 bp. i have already checked my clone with EcoR1 and BamH1. but i want to recheck it with double digestion of one site in vector and one site in insert. but m confused with expected size of bands.
Hi Shubham,
First - cutting with EcoRI will be pointless as there is a SacI site in the vector MCS between the EcoRI and the T overlap. EcoRI/SacI digest will result in a 10bp fragment + your insert fragment (SacI-SacI) and the vector. So you will get the same result (almost) by cutting with SacI only.
Second - your fragment can be inserted in either orientation so there are two possible results from the digest.
Finally - for a SacI digest You should see two bands that are either
3139 (from 3805 (from 2888+971) - 666) + 666 (from 640+26)
or
3502 (from 3805 (from 2888+971) - 303) + 303 (from 277 (from 917-640)+26)
depending upon orientation.
-Richard
p.s. Drawing it out on paper helps.
Hi Richard,
I really appreciate your help. it was great. i always have confusion to understand this concept. can you please suggest me any publication or link to learn or understand this concept.I will be very grateful to you.
Thanks and Regards
Hi Shubham,
I don't know of any publication dedicated to this type of problem. Most cloning textbooks will give some information as to calculating restriction maps. It's really just a logic problem. Many people find it easier if you draw out what you are making on paper using a circle.
Also nowadays there's many DNA manipulation programs that will do all the hard work for you and give you the answer - but might not help you understand what has happened.
-Richard
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