How to perform colony PCR

See more supporting information at www.benchfly.com

Category:

Science & Technology

Tags:

• benchfly
• science videos
• laboratory techniques
• current protocols

[lukceg1983]

I have a question. Can I use for my colony pcr the same primers which I used to amplify this fragment from genomic DNA or is it better to use some primers binding specificaly to the vector backbone?

cheers

lukasz

[benchflydotcom]

@lukceg1983 You should be able to use any primers you want as long as the final product doesn't get too much longer than 2kb. If you've cloned multiple gene fragments into the same vector, you may want to go with vector primers since they would work for all clones and would give you a few extra bases outside the fragment of interest when sequencing. Good luck! -Alan

[Koulouriotikos]

Hello!!! Great job!! I use to boil the colony and centrifuge it before i load it to the master mix. I load 10 μl (no ul) to the mastermix, and then i perfom the pcr reaction. But i will try tomorrow your way. Let's see.

[benchflydotcom]

I've heard of that protocol too.  In my experience, I've never had to boil the colonies first, so it just saves time to skip that step. On our website (see our username for url), we have a text protocol attached to the video, so you can see our recipes. It would be awesome if you would leave your comments over there- I'm sure our users would be very interested in what you have to say! Good luck with your experiment!

[irizarxyz]

Awesome..very useful!! Thanks a lot for the effort..I'm sure it would be very useful if you guys can post lot more of these kind of videos...because reading protocol and performing experiment is not that easy but watching someone do it..definitely helps!!

Good work guys!!!

[benchflydotcom]

Thanks! Check out our site for more videos and information!

[amugoutham]

wonderful !!thanks a lot:)

[VladiSolano]

You guys rocked my bench! Thanks.

[pipo225544009911]

thxxxx!!

[benchflydotcom]

Fantastic - glad it helped! For a written protocol and other videos, check out our website (see our username...). Upload your own on our site or feel free to tell me what you need and we can take care of it!