Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

Table of Contents

Expand All | Contract All

Verification of Cointegrates and Selection of Resolved BAC Clones

(Protocol summary only for purposes of this preview site)

Successful modification of the BAC is confirmed in two separate PCRs. The first reaction (5 cointegrate PCR) uses a forward 5 cointegrate primer (a sequence located upstream of the 5 end of the A-box) and a reverse 3 primer on the vector (175PA50AT) or within the reporter sequence or mutated region as appropriate. The second reaction (3 cointegrate PCR) uses a forward 5 primer on the recA gene (RecA1300S) and a reverse 3 cointegrate primer (a sequence located downstream from the 3 end of the B-box). Those colonies shown to be positive in PCR analysis are further tested for sensitivity to UV light. After the resolution, colonies that have lost the excised recombination vector including sacB and recA genes become UV light sensitive.


Previous   |   Next

Save 30% & Get Free Shipping!

Save 30% at checkout on our website.
(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

Search for information about other protocols included in the book:

Read What Others Are Saying About Molecular Cloning:

Tom Maniatis wins Lasker

'Not Quite a Book Prize'

Get alerts about special offers from Cold Spring Harbor Laboratory Press free to your Inbox