molecular cloning - (Jul/12/2012 )
I am a master student and currently doing my Master thesis in Cloning of keratinase in pichia pastoris. I am very new to molecular biotechnology and i have few doubts. kindly solve these doubts I would be very thankful to all
1.I have a nucleotide sequence of pseudomonas aerugonisa whicg is 1428bp and I am using pPICZ vector which is 3.3 kb. I have order the primer and nucleotide seuencei,what would be the next step that i will be following, will the nucleotide ordered by me will be lyophilzed. i really dont know this is the first time I am dealing with molcular cloning
2.. I would like to know when I double digest the vector and my insert with the ECOR1 and Kpn1, what should I expect as a result, or in otherway can anyone tell me how do I understand restriction digestion comparison with marker. I am unable to understand..
kindly help me out.
The primer will probably arrive lyophilised. You should re-suspend it based on the instructions that come with the primer, this will usually be to 100 uM (stock solution), you will then need to make working solutions - the concentration is usually dependent on your personal preference, but 10 uM is common.
The double digest depends on the vector sequence and the insert sequence - you need to confirm that neither of these REs have a cut site in your insert, (only in the primer sequence) for the cloning. You can look for the independent cutting of the plasmid by each RE by doing each one separately and looking for a single band when run on a gel.
For the double digest, when you run it on a gel, you'll have to run it with a 1kb ladder like the following: http://products.invitrogen.com/ivgn/product/10787018
This will come with a handout of how to interpret your gel.
If you find that your when you load/run your digests on a gel, the digests remain at the top and do not run (thus will appear higher than the ladder) this means the vector DNA has remained circular and did not cut. Otherwise, you can line up the digest products to the ladder to ensure their size.
It might also help to CIP the vector after digesting, which prevents recently digested vectors from closing on themselves, thus rendering them unable to include an insert. http://www.neb.com/nebecomm/products/productM0289.asp
You may not need this but if you find once your plate your ligations that you have a lot of colonies on your control plate, this could help!