route map for cloning a gene - steps involved in subcloning a gene (Oct/11/2007 )
I want to subclone a piece of DNA from a cloning vector to an expression vector. I just want to make sure I have the steps correct and in the right order:
1. Do PCR to amplify DNA
2. Do PCR purification.
3. Do Restriction digestion of the PCR product and vector.
4. Run on agarose gel and gel purify vector and insert
5. Set up ligation
6. Transform into E.coli
7. Screen transformants by PCR or sequencing.
Am I right?
yup more or less, although the last line should be
7. Screen transformants by PCR or restriction digest (miniprep e coli colonies)
8. Grow Midiprep culture and extract plasmid.
9. Check again by restriction digest.
10. If required, check a third time by sequencing.
11. Document. (very important, perhaps more important then the plasmid itself)
The hard part is getting from step 1 to 7.
So best of luck and pray daily... so that the Gods of Molecular biology will look favorably on your bligh...um blessed endevour. Said Gods accept sacrifices of blood, sweat and tears. And chocolate bars are acceptable....though KitKat is preferred. Late nights, tearful pleas, a heart racked by frustration, and coffee are often required to achieve communion with said Gods.
And above all never boast. The Gods of Molecular Biology will punish you!
If you can find compatible restriction sites between the two vector, you don't need to PCR amplify the insert, just cut it and ligate it to the new vector. this will avoid PCR errors and save time and efforts.
For your step 4, you can just purify the digested insert using qiagen PCR purification kit without running a gel.
Be sure to set up different controls during the ligation and transformation steps.
Step 0: Pray that every thing goes fine.