Analysis of recombinant clones from the library

In order to estimate the diversity of the original (i.e. before selection) library, colonies from the ligations are first screened for inserts by PCR followed by digestion with the frequent-cutting enzyme BstNI. Analysis of 48 clones should indicate that at least 90% of the clones have inserts. The BstNI pattern should be very complex indicating that the library is likely to be very diverse. Note though that this is only a quick test and that to really assess the clone diversity, DNA sequencing is a must. 

1 X NEBuffer 2 (see Protocol 10) Agarose (UltraPure) (see Protocol 2) NuSeive 3 1 agarose (25 g, FMC BioProducts, 50091) 

Prepare a bulk solution of the PCR reaction mix (sufficient for 48 clones) by combining the following ingredients  

Using an inoculating needle, gently stab an individual colony from transformation plates taking care not to take too much colony. Twist the needle about three times in the PCR mix in the appropriate well of the microplate. 

Prepare a restriction enzyme mix for BstNl fingerprinting by combining the foUovring ingredients  

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