Team:IBB Pune/Protocols

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[[Image:PROTOCOLS.png|center|700px|center]]
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<span style="font-weight:bold; font-size:150%; color:#6600FF;">Maintenance of microbial cultures</span></html>
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== Maintenance of microbial cultures ==
 
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Standard cultures used in our project are  
Standard cultures used in our project are  
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== Extraction of Genomic DNA ==
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<span style="font-weight:bold; font-size:150%; color:#6600FF;">Extraction of Genomic DNA</span></html>
Extraction of genomic DNA was done by the Chen and Kuo method with some modifications.
Extraction of genomic DNA was done by the Chen and Kuo method with some modifications.
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1) Take 1.5mL of overnight grown culture in a microfuge tube.Centrifuge at 8000rpm for 10 mins.
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[[Image:Klen.png|center|600px|center]]
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2) Decant the supernatant. Resuspend the cell pellet in 100ul resuspension buffer ( 40mM Tris-acetate pH 7,8, 20mM sodium acetate, 1mM EDTA )
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<span style="font-weight:bold; font-size:150%; color:#6600FF;">Extraction of Plasmid DNA</span></html>
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3) Lyse the cells with  400 ul Lysis buffer( 40mM Tris-acetate pH 7,8, 20mM sodium acetate, 1mM EDTA, 1% Sodium Dodecylsulphate)
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4) After complete lysis, add 150ul of 5M NaCl solution. Mix it well. This removes the proteins and the cell debris.
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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.
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6) Add equal volume of chloroform and mix gently by inverting ( around 50 times).
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7) Centrifuge for 15 mins at 10,000 rpm. The aqueous and the organic phases separate out.  
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Extraction of plasmid DNA was done by the Birnboim and Doly (1979)method.The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.
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[[Image:Plasmidisolatn.png|center|700px|center]]
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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.
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<span style="font-weight:bold; font-size:150%; color:#6600FF;">Agarose Gel Electrophoresis</span></html>
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9) RNase treatment is given for 5 mins at RT followed by another chloroform extraction step.
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Agarose Gel Electrophoresis was performed on a horizontal gel apparatus for visualising DNA. Ethidium Bromide was used as the fluorescing dye. EtBr intercalates with the DNA strands and fluoresces under UV light thereby indicating the position of the band
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[[Image:Gelprot.png|center|700px|center]]
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10)Precipitate DNA using 100% isopropanol for 2-3 hrs on ice.
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<span style="font-weight:bold; font-size:150%; color:#6600FF;"><i>in vitro</i> Gene Amplification</span></html>
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11) Centifuge at 12,000rpm for 15 mins, decant the supernatant and dry the DNA pellet in the speed vac.
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Genes to be amplified and identified by Polymerase Chain Reaction (PCR).  
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12) Resuspend the DNA pellet so obtained in 100ul TE buffer.
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[[Image:Pcrfunnel.png|center|500px|center]]
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[[Image:Protocoltable.png|center|400px|center]]

Latest revision as of 03:34, 21 October 2009




PROTOCOLS.png



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.

Klen.png

Extraction of Plasmid DNA

Extraction of plasmid DNA was done by the Birnboim and Doly (1979)method.The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.

Plasmidisolatn.png

Agarose Gel Electrophoresis

Agarose Gel Electrophoresis was performed on a horizontal gel apparatus for visualising DNA. Ethidium Bromide was used as the fluorescing dye. EtBr intercalates with the DNA strands and fluoresces under UV light thereby indicating the position of the band

Gelprot.png

in vitro Gene Amplification

Genes to be amplified and identified by Polymerase Chain Reaction (PCR).

Pcrfunnel.png
Protocoltable.png
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