Lambda Phage Protocol Large DNA Preparation

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Lambda Phage Protocol Large DNA Preparation

Date Added: February 26, 2008 03:54:09 PM

Author: plinkadmin

Category: DNA Protocols

Lambda Phage Large DNA Preparation Day One grow up one colony bacteria (P2 for instance) in 25 mls LB w/ 10mM MgSO4 and 0.2% Maltose autoclave two 400ml aliquots (in 2-liter flasks) of RPM, when cooled add Maltose to 0.2% Day Two - Large Lambda Phage DNA Prep to each of two 10 ml aliquots of bacteria (from above) add 10^7-10^8 phage (from a liquid lysate) incubate in culture tubes at 37oC for 20 min. add the bacteria/phage to each of the 400 mls of media (from above) and shake at 300 rpm,37oC until lysis begins (approx. 6-8 hrs but depends on the bacteria/phage ratio;could take as long as 12 hours) --it is very important to capture this when lysis begins add 5 mls chloroform to each and continue shaking for 15-30 min. --the chloroform should lyse all cells resulting in release of phage heads, however if the cells had been allowed to finish lysing on their own, the released phage would have had time to attach onto pieces of cell membrane, injecting their DNA into the solution where it could not be recovered add 23.5 g NaCl to each, dissolve by swirling, let sit in cold room for 15 min. centrifuge at 10,000 g for 10 min, 4oC to get rid of cellular debris. collect supernatant (at this point you can let it sit in the cold room overnight or go on) add PEG (10% weight/volume supernatant) and stir slowly in cold room 1-2 hrs. spin at 8000 g for 20 min. resuspend pellet/smear in 10 ml SM (5 to resuspend, 5 to wash sides of container) add 55 ug DNase, 50 ug RNase, and let sit at room temp for 20 min. add 7.8 g CsCl and swirl gently until dissolved transfer to seal-a-tube and spin in ultracentrifuge for 20 hr., 50K, 4oC or 24 hr,45K, 4oC identify white band of phage heads and use a needle and syringe to capture this band (before inserting needle at band site, puncture a hole at the top of the tube) dialyze against a large volume of SM at least 4 hrs, changing SM twice.

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