hello
i want to ask that which enzyme i best to chose for restriction digestion of insert (2Kb) and vector (pCAMBIA 3301) like i want to use NcoI and BstEII which are located in the position of GUS reporter gene if i use this enzyme then which on is the reporter gene?
and if i use any enzyme for digestion do i need to add some Base pairs of protection sequence before the restriction enzyme sequence and tell me how much bp of protection site is suitable and from where i get information about which protection site is suitable for which enzyme.?
for example:
for my primer (to amplify cDS ) i add restriction enzyme here shown bold and itelic, protection site six bp before enzyme and `+` is my primer sequance.
Farward primer = TCTTGACCATGG`5+++++++++++++++++++++++++3`
Reverse primer = TTACAGGTGACC`5+++++++++++++++++++++++++3`
Your best choice would be a restriction enzyme within the multiple cloning sequence (MCS) e.g. EcoRI and HindIII. This will give you a number of advantages:
- MCS is within the lacz gene. This means you can use blue-white selection in E. coli.
- You will keep the GUS reporter.
- You will keep the His tag, which you will need if you intend to purify your protein.
As for the number of nucleotides required before the restriction site, you can consult NEB webpage for this purpose (https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments). Generally, it's safe to include 6 adenine bases before the restriction site to ensure efficient cleavage. the nature of the nucleotides is not really important because they will be cleaved anyway; just make sure they don't form secondary structures e.g. hairpins to avoid reduction in PCR efficiency.
thanks brother Ali for your time i appreciate you answer.the question which arise now is like i need the promoter CAMV 35s. and so if i will use the MCS then i need to add some more critical steps to add in my experiments which i don`t want to do so in that situation whats you kind suggestion for me.
whats the problem if i replace the gus?
In this case, I think the best choice would be NcoI and BglII, which will insert your gene upstream of GUS reporter. Please, make sure to do the following during primer design:
- make sure that your insert is in-frame.
- make up for the lost nuclotides from the GUS reporter to avoid the loss of MVDL from N-terminal. Please, avoid recreating the restriction sites once again in this step. Use synonymous codons to create the same amino acids.
I really recommend using SnapGene.
With regards to GUS, it helps you monitor the expression of your gene under the promoter CAMV 35s in a histochemical way (https://en.wikipedia.org/wiki/GUS_reporter_system). If you remove it, you will lose this advantage.
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