Hi,
I am going to prepare Genomic DNA library for NGS, I have cultured gut bacteria on various media and scrap the plates,I use tiangen kit to isolate DNA but it is shared and for Genomic DNA library it can,t work, my Question is what can be the best protocol to isolate DNA, The culture plates contains almost 700-1000 bacterial colony of both gram positive and gram negative bacteria.
Hi,
I used Invitrogen ChargeSwitch gDNA Mini Bacteria Kit (CS11301) for my C. difficile WGS research. There are two extraction protocols for G+ and G-.
Only what did notwork in my case was library preparation with DNA extracted form C. difficile culture in selective media. DNA extraction from non selective media works great.
Good luck.
Hello Mati,
you have several options:
1) You can use a commercial extraction kit (eg Qiagen) which will give you very clean DNA and the yield will be quite good.
2) You use phenol-chloroform extraction followed by alcohol precipitation which will give you a higher yield although the DNA solution might still contain residues of phenol-chloroform.
3) You use mechanical disruption with ceramic beads. But for this you will need a high-speed shaking device (Precellys).
What will improve your DNA yield anyway is to use a preincubation step with an enzyme in TE.
For gram negative bacteria you can use proteinase K (10mg/ml) at 56°C for 1hour. Adding SDS at 1% increases the disruption of cells.
For gram positive bacteria you should use lysostaphin at 37°C for 1 hour.
Best regards
Dirk
Rose method. Rapid one step extraction method, this method is best method for dna extraction from bacteria
for high quality and good DNA extraction, there are many commercial different kits can be used, for higher yield u can use manual DNA extraction protocol like salting out or phenol/chloroform.
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