Cosmid library-Large -scale prep of cosmids

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
Take one isolated colony and transfer it to a starter of 10 ml LB +the proper antibiotic and let it grow for 6-8 hours.
Transfer the strater to 1 liter LB +antibiotic and let it grow over night at 37 C in shaker.
Centrifuge 15 minutes at 4000 rpm
Resuspend the pellet in 20 ml of solution I
(50mM D-glucose, 25mM Tris-HCl,10mM EDTA). Pass to 250 ml tubes.
Incubation 5 minutes on ice.
From here use only plastic pipetes!
Add 50 ml fresh solution II (0.2N NaOH,1%SDS ), mix gently
Incubation 5 minutes on ice. (no more!)
Add 37.5 ml solution III (8M Am4Ac), mix gently until white flakes uppear.
10 minutes incubation on ice.
Centifuge 30 minutes in GSA rotor on 10000 rpm.
Filter the supernatant through 4 layers of gaza to new tubes.
Add 0.6 volume of isopropanol (70 ml)
Mix and incubate 20 minutes at room temp. (Its better to longer incubation until 1 hour)
Centrifuge in GSA in 10000 rpm for 20 minutes.
Keep the pellet and wipe the entrance of the tube (there is also pellet on the wall which is hard to see). Resuspend the pellet in 4 ml DDW. Transfer to polyproplen tubes and see the pellet is well resuspended.
Add 8M Am4Ac and mix gently for 1 minute.
Incubate 10 minutes on ice.
Centrifuge in HB-4 rotor 10 minutes in 8000 rpm.
Transfer the supe to new tubes
Add 6 ml isopropanol and mix gently. (It's possible to stop here over night in -20 C.) Or to incubate 15 minutes at room temp.
Centrifuge for 10 minutes in 8000 rpm.
Keep pellet.
Wash with 70% Ethanol: Fill up the tube, centrifuge until 5000 rpm and throw all the supe.
Resuspend in 1 ml DDW
Add 20 ml RNAase A from 5 mg/ml. Incubate for 30 minutes at 37 C.
Add 580 ml DDW.
Add 400 ml of 4M NaCl. Mix gently.
Add 2 ml 13 % PEG 8000. Mix gently.
Incubate 1 hour on ice.
Centrifuge 20 minutes at 80000 rpm.
Keep pellet and wash in 70% ethanol as described before.
Dry tubes and resuspend well in 3.3 ml DDW.
Add 1.7 ml of 8M Am4Ac and mix gently.
Phenol/Chloroform/Isoamyl. Centrifuge in table centrifuge for 5 min.
Repeat step 34 again if not clean.
Chlorophorm/Isoamyl twice
Add 2.5 volume of 100% Ethanol
Centrifuge 30' in 10000 rpm
Wash with 70 % ethanol as before
Dry and resuspend the DNA in 200 ml DDW.

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Cosmid library-Large -scale prep of cosmids