Single Strand DNA Preparation Background
Single stranded DNA consists of one chain of nucleotides rather than the two base pairing strands found in the double stranded DNA form. The family of viruses belonging to parvoviridae have a single stranded DNA genome. In DNA replication and in the copying of RNA from DNA, the strands of the helix must seperate at least temporaily. During DNA synthesis two new strands are made (one copied from each of the original strands), resulting in two double helices identical with the original one. In the case of copying the DNA template to make RNA, the RNA is released and the two DNA strands reassociate with each other.
Single stranded DNA can be prepared by heating DNA enough to break the energy of the hydrogen bonds. Because DNA has the tendency to reanneal instead of staying single stranded it is cooled rapidly on ice instead of slowly cooling it which will cause reannealing.
Heating DNA causes the strands to separate and rapid cooling prevents renaturation.
Single Strand DNA Isolation Protocol
20% PEG/2.5M NaCL
for 100 mls
PEG (polyethylene glycol,MW 8000): 20g
20 gramsNaCl : 14.6g
0.3 M NaOAc/1mM EDTA
for 100 mls
0.3M NaOAc: 10 ml of 3M
1mM EDTA: 200 ul of 0.5M
- Inoculate a single colony into 5ml of SB (2xYT) containing 100 ug/ml AMP and 10 ul M13KO7 helper phage at 107 -108 pfu/ul
- Grow the culture at 37oC with vigourous aeration for 1.5 to 2 hrs.
- Add kanamycin to 70ug/ml to select for phage infected cells.
- Continue to grow at 37oC for 16 to 24 hrs or until growth is saturated.
- Centrifuge 5 mls of cells for 5 min at 15k g (10500 rpm)
- Remove 5 ml of super and add 750 ul of ice cold 20% PEG/2.5M NaCl. Allow phage to preciptate on ice 1 hr.
(Save pellet for double strand prep.)
- To supernatant with PEG., centrifuge 10 min. at 15k g (10500 rpm) a pellet should be obvious. (phallic smear- that's what it looks like!!)
- Remove and discard the supernatant. Centrifuge the PEG pellet a few seconds more to collect residual liquid and remove. REMOVE ALL LIQUID.
- Resuspend the pellet in 400 ul of 0.3M NaOAc pH 6.0/ 1mM EDTA by votexing vigourously. (MAKE SURE PELLET IS IN SOLUTION!!!)
- Phenol/CHCl3 (chloroform) extract, CHCl3 extract.
- To supernatant (top phase) in a fresh tube, add 1 ml EtOH, -20oC 20 min, Centrifuge 30 min.
- Remove EtOH, wash with 70% EtOH, dry.
- Dissolve in 25 ul of T.E. Run 2 ul on agarose gel. Use 7 ul for each sequencing reaction.