NdeI restriction site was added in forward primer and HindIII was added in reverse primer size of band is looking near 500 bp but size of is less than 15 kda but expected size should be 17.7kda.
Are both restriction sites still present in the clone?
Also did you sequence more than one plasmid, you may have just picked a bad clone.
However if multiple clones look like this, then I would inspect your primers and your sequence carefully to see if you can identify something strange, since you have the precise junction points from your sequence you might be able to deduce what happened.
maybe you have a nde1 or hindIII site IN your PCR fragment... how are the junctions between your insert and vector?
NdeI and HindIII is zero cutter for amplified PCR product. and it is single cutter for pet30a(+) vector. this, I checked from NEB 2.0 wabsite.
maybe you have a cryptic site in your insert that could be cleaved if you add too much enzymes or if you are not in the right buffer; are hindIII and ndeI working in the same buffer? did you get a lot of clones? did you analysed several of them ? by sequencing or by restiction? are the Nde1 and HindIII effectively present in your clone? where is the deletion on your sequence?
Asper discussion, I choosed the different clone of 1:1 ration fraction, I got the size of purified protein near to the expected size 17.7kda and very thick band but there was also thin nonspecfic protein band saw in Western blot result whose size is more than 20kda but less than 23 kda but size of band is very thin. can anyone please tell me ?
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