Introduction
The aim of this protocol is to describe the sub-cloning procedure Ð the techniques used to take a DNA fragment from one plasmid and transfer it to another.
Timetable
There are several step to Sub-cloning, which can be performed in one day, but requires good time management.
1. Set-up RE digest and incubate for at least 60 mins
2. SAP treat the vector (NOT the insert)
3. Gel purify fragments and ligate (for blunt end ligations, overnight incubations are necessary)
4. Transform ligated DNA into E.coli
Aim
The example that you will perform for this part of the course requires the subcloning of P2X2 ORF from the TA-TOPO vector into pEGFP-N1 (Clontech Ltd.) to create a P2X2-EGFP fusion protein.
A P2Y-GFP fusion protein has been described previously (Vohringer, C. et al. (2000). A chimeric rat brain P2Y(1) receptor tagged with green-fluorescent protein: High-affinity ligand recognition of adenosine diphosphates and triphosphates and selectivity identical to that of the wild-type receptor. Biochemical Pharmacology 59, 791-800).
You will isolate a 1.4kb PstI-BamHI DNA fragment (insert) containing the amplified P2X2 sequence (that you generated using protocols #4 - #7) and clone it into the PstI-BamHI digested backbone fragment of pEGFP-N1 (vector). Note, that the vector is also treated with an additional enzyme, shrimp alkaline phosphatase (SAP).
Figure Cartoon representation of sub-cloning strategy
PstI and BamHI recognition sequences are:
