UV Lab/ Protocols/15
Mutagenesis
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1. Dilute the over night culture of E. coli in L.B and aerate at 37 C till mid llog phase(3 hrs.).
2. Centrifuge 2 ml. of culture and wash twice with 2 ml. saline and resuspend in 2 ml. of Tris
malate buffer.
3. Serially dilute and plate for obtaining initial cell no.,. Plate 0.1 ml. of each culture on
selective plates for testing spontaneous mutation frequency.
4. Add a freshly prepared solution of MNNG (1 mg./ml. in saline ) to give 50 µ g /,.
Keep at
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37 C for 20 min.
5. Draw asample at the end of the reaction and serially dilute in saline.Dilute and plate for
scoring survivors.
6.Centrifuge the treated cells and wash thrice with saline to remove MNNG as much as
possible.
7. Resuspend the treated cells in 10 ml. of L.B and let grow O/N at 37 oC.
Day 2:
Serially dilute the culture upto 10 -6. Plate 0.1 ml. of 10 0, 10 -1, 10 -2 dilutin on selective
media plates.
Tris - Maleate Buffer for 100 ml.
Maleic acid
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0.508 g.
Tris base
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0.605 g.
H
Dissolve the both on water. adjust the p to 6.5 , make it upto 100 ml.
Time points:
0,5,10,20,30,40