Analysis of Viral ssDNA by Agarose Gel Electrophoresis

Analysis of Viral ssDNA by Agarose Gel Electrophoresis

There are multiple reasons to analyze the closed circular ssDNA in filamentous phage preparations; it is a straightforward way to evaluate the size of the phage genome and detect the presence of contaminants that may hinder phage display; presence of insert and deletions can also be detected in some cases (Fig. 1). This protocol is derived from the laboratory manuscript “Cloning in fUSE vectors” by George Smith (1).

Figure 1. ssDNA analysis of a pADL-10b/scFv clone rescued by M13KO7.

Material & Reagents

GBB Buffer (40x stock)

This is the electrophoresis buffer. It has a high conductivity and voltage must be kept low to prevent melting of the agarose.

For 200 ml:

 

Tris base

40.7 g
 

NaOAc anhydrous

13.1 g
 

Na2EDTA.2H2O

5.38 g
 

Dissolve in ~ 150 ml of water;
Adjust pH to 8.3 with glacial acetic acid;
Adjust final volume to 200 ml;
Store non-sterilely at RT.

Lysis mix (2x solution)

This is the phage lysis buffer.

For 5 ml:

 

SDS 2% w/v final

1 ml solution at 10% w/v in water
 

Glycerol 20% v/v final

2 ml solution at 50% v/v in water
 

GBB 8x final

1 ml 40x stock solution
 

Bromophenol blue

2 mg (“the tip of a spatula”)
 

Water

1 ml

Procedure

  1. Preheat Lysis mix solution at 42ºC or more before use.

  2. Mix an equal volume of 2x Lysis mix and virion preparation around 1×1013 virions/ml (around 1 OD for most phagemids).

  3. Heat for 10 min at 50ºC-70ºC.

  4. Load 10 µl per lane on a 1.5% agarose minigel in 4x GBB buffer containing ethydium bromide.

  5. Load a DNA ladder on first well; phage ssDNA runs usually between the 2 kb and 3 kb markers.

  6. Run for a few hours at 20-30 V; higher voltages may result in melting of the agarose.

  7. Analyze under U.V. using your favorite gel documentation system.

References

  1. Smith G. (1992). Cloning in fUSE vectors; edition of February 10, 1992, University of Missouri, Columbia.

 

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