Colony PCR for clone screen
Posted 18 January 2005 - 02:57 AM
Posted 18 January 2005 - 03:14 AM
not sure i understand your question...
before sending for sequencing it is usually advisable to check if your plasmid has the right-sized insert in it, and this can be done either by extracting the plasmid and cutting out the insert, or by PCR . Doing colony PCR is usually quickest and if you get a good unique band, then you can directly sequence the product without needing to extract plasmids at all.
in many cases it helps to dilute the colony in 50 - 100microliter ddw, and then take 1 microliter as template per reaction.
Posted 19 January 2005 - 01:17 AM
If you want to check sequence of your recombinant plasmid (contain new insert), you should do colony PCR first.
Posted 05 March 2005 - 09:09 AM
For over 20 colonies, I pick and patch, and screen via colony hybridization.
For colony PCR, you should be able to simply touch a sterile toothpick to your colony, and then dip it into your pcr solution. Always use a black as colony pcr is the only time I have ever had contamination.
Posted 07 March 2005 - 09:39 AM
I routinely do PCR colony screening, to determine positive clones, number of inserts and direction of inserts, especially if problems with background re-ligation. As far as is it necessary if you only have a few colonies with a single insert than just mini-prep, if it is anything more complex than use do colony screen.
Hope this has helped.