Hi Damien,

have a look at Phage Lambda Genomic libraries & cDNA libraries.

Once the correct clone was found by hybridisation with a radio-labled probe, the DNA could be cloned using classical cloning techniques, along the lines you have outlined.

-Richard

https://en.wikipedia.org/wiki/Genomic_library

https://en.wikipedia.org/wiki/CDNA_library

OHHHH yeahh, hybridisation! Hm.. if I used the technique above, I would get many colonies each with different and random inserts, right?

Hi Damien

in essence yes. This is how persons like myself made constructs in the 80s.  I would add one or two more points to your procedural pipeline: 

1.  Having digested your plasmid and then purified ( back then by phenol chloroform extraction and ethanol precipitation) it was common practice to treat it with calf intestinal phosphatase ( CIP). This cipping procedure as it was known dephosphorylates the sticky restriction ends of your linearised parent vector and forces it to recombine through the insert ( which provide terminal phosphates). Typically as I recall I would treat 1ug of purified cut vector with 1ul of CIP at 37C for 15min and then proceed with overnight ligation at 16c using T4 ligase.  This procedure obviously increases the efficiency of 'correct' ligation

2.  In terms of ligation it was considered good practice to have at least 3 molar equivalents of your purified insert to 1 molar equivalent of your Cipped purified cut vector.  In essence add as much insert as you can to your vector to encourage recombination via your insert

3.  Having transformed and played preferably perform blue/ white selection with xgal and IPTG ( which you add to your LB agar):  look up commercial methods for explanation and protocols.  In essence true recombinant colonies are white and parent re lighted colonies remain blue  this enables you to pick colonies selectively and increase your chances of obtaining your correct clone

4.  Whether you use blue white selection or not pick 6-12 colonies for mini prep and digest examination.  With such a number I would typically find 1-5 were what I wanted ( the larger number if I had used blue white selection)

5. Finally when playing out include a mock transformants where buffer had been added instead of purified and insert and transformation had been procured in a second control with insert alone:  the following day if everything worked correctly I would typically get 1 or 2 colonies on my insert only plate; 20 or so colonies on my Cipped vector only plate and perhaps 100 on my Cipped parent vector plus insert transformation mix indicating that recombination was most efficiently obtained via the insert;  that is a large proportion of my colonies were likely to be the correct construct 

Hi there

i have provided links to the CIP enzyme and principle of blue white colony selection if you decide to do thst

i will just add that when I cloned this using this old fashioned method I would

1. Digest 1ug of parent plasmid

2. Check that digestion was complete by running 1/10 if what I had digested on an agarose gel

3. Purify remainder of digestion mix ethanol precipitate then treat with 1ul of CIP at 37C for 15min then re purify 

4. Digest my insert and purify

5. Add to the CIP treated parent vector in a minimum ratio of 1:3

6. Ligate overnight at 16C with T4 DNA ligase 

7. Transform then make up to 1ml with LB ( as per standards protocol)

8.  Plate out 50ul and 250ul of this mix using the combinations I talked about above : typically those colony numbers were found on the 250ul plate 

https://www.neb.com/products/m0290-alkaline-phosphatase-calf-intestinal-cip

Blue white selection

http://www.sigmaaldrich.com/technical-documents/articles/biology/blue-white-screening.html