Cosmid library-Cosmid library screen using PCR

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
Since colony hybridization on cosmids tends to give a lot of background, a PCR screen is preferable. Also dotblots gave bad results.
Design primers and check first on total genomic DNA of L. pennellii if a strong band is detected. Consider sending the amplified band of for sequencing to make sure it is the desired sequence you are amplifying.

Do standard PCR on all 96 superpools of the library, use 1 ml per pool as template. Include as positive control 1 ml L. pennellii DNA and as negative control 1 ml water. Analyze PCR on gel and choose the pools showing the same fragment as the positive control. You should expect around 3 to 4 pools to be positive.

Grown 3ml LB/5tet overnight cultures, at 37 C, of each of the pools. Do isolation of DNA (miniprep) and repeat PCR. If bands are observed go ahead and start screening.
DNA isolation:

2 ml culture in eppendorf, spin 6500 rpm, 5 min, RT
resuspend pellet in 200 ml 25mM Tris, 50 mM glucose, 10 mM EDTA (pH8), 5 min RT
add 400 ml fresh 0.2N NaOH/1%SDS, invert several times, 5 min in ice
add 300 ml 8M NH4Ac, mix gently, 10 min on ice
spin 10 min, 14000rpm, RT, transfer SN to fresh tube
add 0.6x vol (550ml) isopropanol, 10 min RT
remove SN, wash pellet with 1 ml 70% ethanol
spin 5 min 14000rpm, RT
dry pellet 15 min on bench
resuspend in 50 ml water

Titer the pools to calculate how much is necessary for around 4000 colonies per big plate (150 mm). Take from glycerol stock 20 ml bacteria and dilute into 200 ml LB. Plate 10 ml (=1x) on a 90 mm plate containing LB and 5 mg/ml tetracyclin and incubate overnight at 37 C. Plate also 10 ml 0.1x , 10 ml 0.01x and 10 ml 0.001x. Typically the titer comes out such that you nedd to spread around 50 ml of the 0.01x to get 4000 clonies on a big plate

Spread for each positive pool bacteria on big plates LB/tet to get 4000 colonies per plate. Do also 1 concentration higher and lower, just as back up. Grow overnigt at 37 C

Cut the plate containing 4000 colonies into 4 and harvest colonies from each piece seperately into 1 ml of water, by scraping with a sterile scalpel (each tube now represents 1000 colonies, and receives an individual number, p.e if positive superpool is number 24, the 4 quarters will be 24.1, 24.2, 24.3, 24.4). Do miniprep DNA on 950 ml as described above and PCR. Don't forget controls! Keep the last 50 ml at 4 C to spread for the next round. Run 10 ml of the PCR reaction on gel and look for which quarter has a positive band (p.e. 24.1). This is the one you will chose for the next round

Of the positive quarter (24.1) take the left over 50 ml and dilute 10 ml in 990 ml (=0.01x), of this take 10 ml into 90 ml (=0.001x) and of this one again take 10 ml into 90 ml (=0.0001x). Spread of all 3 dilutions 25 ml per big plate LB/tet5. grow overnight at 37 C.

Choose the plate dilution that has around 1000 colonies per plate and cut into 8 (24.1.1, 24.1.2, 24.1.3 .... 24.1.8). Each piece now represents around 125 colonies. Harvest as above into 1 ml water. Keep 50 ml seperate and isolate DNA from the 950 ml. Do PCR and analyze on gel. Identify the positive one (p.e. 24.1.3) and spread again at 3 concentrations on LB/tet5.

Choose the plate dilution that has around 125 colonies per plate and cut into 8 (24.1.3.1, 24.1.3.2, 24.1.3.3 .... 24.1.3.8). Each piece now represents around 15 colonies. Harvest as above into 1 ml water. Keep 50 ml seperate and isolate DNA from the 950 ml. Do PCR and analyze on gel. Identify the positive one (p.e. 24.1.3.5) and spread again at 3 concentrations on LB/tet5.

Choose the plate dilution that has around 50 colonies per plate. Do individual liquid cultures, 3 ml LB/tet5 overnight 37 C, of 30 to 50 of the colonies. (24.1.3.5.1, 24.1.3.5.2, 24.1.3.5.3 .... 24.1.3.5.30). Next day, do DNA miniprep of 2 ml, keep 1 ml aside at 4 C. Do PCR and identify positive colony. Make glycerol stock. You have isolated your cosmid! Try to have at least 2 independent cosmids (i.e. from seperate superpools).

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Cosmid library-Cosmid library screen using PCR