Team:IBB Pune/Protocols

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Revision as of 05:32, 27 September 2009

Maintenance of microbial cultures

Standard cultures used in our project are

1) E.coli DH5a

2) E.coli JM101

3) Acinetobacter baylyi BD413

4) Streptococcus pneumoniae R6

Glycerol stocks of all the strains are maintained at -80oC.

Strains are maintained on working plates for daily use, subcultured every week. Working plates used are Luria Bertani Agar for E.coli and Streptococcus strains and CLED (Cysteine Lactose Electrolyte Deficient) Agar for Acinetobactersp.

Extraction of Genomic DNA

Extraction of genomic DNA was done by the Chen and Kuo method with some modifications.

1) Take 1.5mL of overnight grown culture in a microfuge tube.Centrifuge at 8000rpm for 10 mins.

2) Decant the supernatant. Resuspend the cell pellet in 100ul resuspension buffer ( 40mM Tris-acetate pH 7,8, 20mM sodium acetate, 1mM EDTA )

3) Lyse the cells with 400 ul Lysis buffer( 40mM Tris-acetate pH 7,8, 20mM sodium acetate, 1mM EDTA, 1% Sodium Dodecylsulphate)

4) After complete lysis, add 150ul of 5M NaCl solution. Mix it well. This removes the proteins and the cell debris.

5) Centrifuge at 12,000rpm for 10 mins. All the cell debris and the proteins settle down. Transfer the clear supernatant to a new microfuge tube.

6) Add equal volume of chloroform and mix gently by inverting ( around 50 times).

7) Centrifuge for 15 mins at 10,000 rpm. The aqueous and the organic phases separate out.

8) Genomic DNA is present in the aqueous phase i.e. the top phase. Tranfer into a new microfuge tube. The interphase contains the proteins. You may repeat this step twice to maximally reduce protein contamination.

9) RNase treatment is given for 5 mins at RT followed by another chloroform extraction step.

10)Precipitate DNA using 100% isopropanol for 2-3 hrs on ice.

11) Centifuge at 12,000rpm for 15 mins, decant the supernatant and dry the DNA pellet in the speed vac.

@@ Line 4: / Line 4: @@
 == Maintenance of microbial cultures ==
-  Standard cultures use in out project are
+Standard cultures used in our project are
 ) ''E.coli DH5a''
 ) ''E.coli JM101''
 ) ''Acinetobacter baylyi BD413''
 ) ''Streptococcus pneumoniae R6''
 Glycerol stocks of all the strains are maintained at -80oC.
-Strains are maintained on working plates for daily use, subcultured every week. Working plates use are Luria Bertani Agar for ''E.coli'' and ''Sterptococcus''strains and CLED (Cysteine Lactose Electrolyte Deficient) Agar for ''Acinetobacter''sp.
+Strains are maintained on working plates for daily use, subcultured every week. Working plates used are Luria Bertani Agar for ''E.coli'' and ''Streptococcus'' strains and CLED (Cysteine Lactose Electrolyte Deficient) Agar for ''Acinetobacter''sp.
+== Extraction of Genomic DNA ==
+Extraction of genomic DNA was done by the Chen and Kuo method with some modifications.
+) Take 1.5mL of overnight grown culture in a microfuge tube.Centrifuge at 8000rpm for 10 mins.
+) Decant the supernatant. Resuspend the cell pellet in 100ul resuspension buffer ( 40mM Tris-acetate pH 7,8, 20mM sodium acetate, 1mM EDTA )
+) Lyse the cells with  400 ul Lysis buffer( 40mM Tris-acetate pH 7,8, 20mM sodium acetate, 1mM EDTA, 1% Sodium Dodecylsulphate)
+) After complete lysis, add 150ul of 5M NaCl solution. Mix it well. This removes the proteins and the cell debris.
+) Centrifuge at 12,000rpm for 10 mins. All the cell debris and the proteins settle down. Transfer the clear supernatant to a new microfuge tube.
+) Add equal volume of chloroform and mix gently by inverting ( around 50 times).
+) Centrifuge for 15 mins at 10,000 rpm. The aqueous and the organic phases separate out.
+) Genomic DNA is present in the aqueous phase i.e. the top phase. Tranfer into a new microfuge tube. The interphase contains the proteins. You may repeat this step twice to maximally reduce protein contamination.
+) RNase treatment is given for 5 mins at RT followed by another chloroform extraction step.
+)Precipitate DNA using 100% isopropanol for 2-3 hrs on ice.
+) Centifuge at 12,000rpm for 15 mins, decant the supernatant and dry the DNA pellet in the speed vac.