Genomic DNA Preps
RGB12955%USA.decnet at USAV01.GLAXO.COM
Tue Nov 3 13:22:00 EST 1992
Thanks to all who replied to my query for Genomic DNA Prep information. Of the
four replies, two included protocols that may be suitable for my non-molecular
biologist friends. The others suggested that nuclear preps of trypsinized (not
scraped) cells would make life easier for subsequent manipulations in general.
Glaxo Research Institute
>Place a SMALL number of cells in 200 ul of DDW containing 5- 10% of CHELEX-100
>resin (Tech. grd. from Bio-Rad). Boil the sample for 20 min's. spin down well,
>and take an aliquote of 10-15 ul from the upper surface of the sup.
>PCR from Whole Cell Lysates
>1. Pellet cells and resuspend in 1X PBS at a concentration of 2 x 10^6
>2. Add 4-8ul of cells (8,000-16,000 cells) to a 0.5ml eppidorf.
>3. Add H2O to 20ul.
>4. Heat at 90oC for 10 min to lyse cells.
>5. Spin 15 seconds in a microcentrifuge.
>6. Add 1ul proteinase K (10mg/ml in H2O).
>7. Incubate at 55oC for 30 minutes.
>8. Spin 15 seconds in a microcentrifuge.
>9. Heat at 90oC for 10 minutes to inactivate the proteinase K.
>10. Spin 15 seconds in a microcentrifuge.
>11. Heat to 94oC for 1 minute and then add 29ul of PCR mix containing buffer,
>12. Cycle 35X at appropriate temperatures.
Note: This protocol gave very good results for an 871bp product from the
>If you're bent on getting a kit, I would recommend either Bio101 (tele
>#1-800-424-6101) G NOME DNA kit which is nice in that you don't have to phenol
>extract, otherwise Schleicher and schuell makes a kit which is easy to use and
>gives clean samples although at a somewhat reduced yield in my hands.
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