Protocol S.2

Single Strand DNA Prep. for Sequencing

This protocol works well from a 5 ml starting culture or a 1 ml starting culture (adjust volume accordingly).

 

Solutions

2X YT Media

16 g tryptone

10 g yeast extract

5 g NaCl

1 ml 1N NaOH

up to 1 liter with Q

Ampicillin Stock (1000X)

0.15 g ampicillin

1 ml Q

can be stored at 4° for several weeks

Tetracycline Stock (1000X)

15 mg tetracycline

500 ml EtOH

500 ml Q

vortex to dissolve and store at 4°

Kanamycin Stock (1000X)

50 mg kanamycin

1 ml Q

can be stored at 4° for several weeks

 

 

20% PEG 8000/ 2.5 M NaCl

20 g PEG 8000

14.6 g NaCl

up to 100 ml with Q

Procedure

• Transform the appropriate plasmid construct into XL-1 Blue and plug one colony into 5 ml 2X YT + Amp + Tet.

• Add 10 ml Helper phage (M13 VCS 1x1012 pfu/ml) and incubate in the 37° shaker for 45 minutes.

• Add kanamycin and continue to incubate in the 37° shaker overnight.

• Pellet the cells at 4° for 10 minutes and transfer 1.2 ml of supernate to each of 4 eppendorf tubes.

• Discard the cell pellets and add 240 ml 20% PEG/2.5 M NaCl to each eppendorf tube.

• Incubate on ice for 30 minutes and spin at 4° for 15 minutes to pellet the phage.

• Aspirate the supernate and resuspend the pellet in 400 ml Q.

• Phenol/CHCl3 extract and CHCl3 extract.

• Add 0.1 volume of 3 M NaOAc, 1 ml Glycogen and 2 volumes of EtOH.

Precipitate, wash and dry and resuspend in 50 ml Q.

• 5-7 ml is generally enough for each sequencing reaction.