Cosmid library-Cosmids pools screening

Thursday, December 04, 2003

Description
The library is a L. pennellii library. The cosmid library was made from MboI partially digested total genomic DNA which was size fractionated on a sucrose gradient and selected for a range of approx. 15-30 Kb. the DNA was ligated into the cosmid vector T04541. This vector is a binary vector with left and right border sequences, a NPTII gene for selction in plants. The vector has a tet marker for selction in bacteria.

Procedure
Fixing the titer
Dilutions were made in LB from 10-1 to 10-6. From each dilution 10 ml were plated on 90mm tet plate..Plates were grown over night at 37 C.Colonies were count.The dilution which gave by calculation about 4000 colonies in 140mm plate was chosen as the dilution which should be used.

Plating the bacteria
According to the titer the bacteria were plated on 140mm LB-tet plate and were grown in an inverted position for 12-14 hours at 37 C. Plates that are 2-3 days old give better results because they absorb the inoculum more readily.
Plates were chilled for 30-60 minutes at 4 C.

Replicating colonies onto nitrocellulose filters
Filters were numbered with a soft lead pencil according to the plates.
Plates were marked in three or more asymetric locations by stabbing them with needle attached to a syringe containing waterproof black drawing ink.
The filters were placed numbered side down on the plates,in contact with the bacterial colonies,until it is completely wet for 2 minutes.
The filters were peeled off and were placed colony side up on 1ml denaturating solution (0.5 N NaoH,1.5 M NaCl) on a piece of Saran Wrap for 2 minutes.Excess liquid was poured off on 3MM paper .
For each filter step 4 was repeated using a fresh Saran Wrap and fresh denaturating solution.
Each Filter was then transfered to 1 ml Neutralizing solution (1.5 M NaCl,0.5 M Tris (ph=7.4)), on a fresh piece of Saran Wrap for 2 minutes.Execess of liquid was poured off on 3 MM paper.
For each filter step 6 was repeated using a fresh Saran Wrap and fresh neutralizing solution.
Filters were transfered to 2X SSC solution for 5 minutes
Filters were layed ,colony side up ,on a sheet of dry 3MM paper and were allowed to dry at room temperature for at least 30 minutes.
The master plates were incubated for 5-7 hours at 37 C until the colonies have regenerated.
Lifting filters was done once more exactly as described,for duplicates in the results.
Step 10 was repeated ,then the plates were sealed with Parafilm,and stored at 4 C, in an inverted position.

Hybridization
Filters were hybridized to a p32 labeled probe, washed, and exposed to x-ray film as described in maniantis.

Reading the results
The films were align with the filters using the phospholabeling marks. The films were marked by pen with the positions of the asymmetrically located dots on the numbered filters. Positions of positive hybridization signals were marked as well.Identification of positive colonies was made by aligning the dots on the film with those on the agar plate.

Second screening
The positive colonies were picked up by the opposite side of paster pippete with the agar and were grown in 2-3 ml LB + the proper antibiotic for several hours at
37 C.Often,the alignment of the filters with the plate does not permit identification of an individual hybridization colony.In this case,several adjacent colonies were pooled. Three dilutions of 10 -1,10-2,10-3 were plated on 90mm LB plates, 10 ml from each dilution. Plates were grown at 37 C over night. Colonies were plated again in isolated way on LB plate which contains 50 isolated places for 50 colonies.The more adjacent hybridization colonies were picked the more colonies were replated for second screening. Plates were grown at 37 C over night. These colonies were then screened a second time by hybridization,as the same as the first screening. A single,well isolated positive colony was picked up from the second screening and used for further analysis.

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Cosmid library-Cosmids pools screening