Genomic DNA Preps

USA::RGB12955 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.

Rich Buckholz
Glaxo Research Institute

The protocols:

>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.

or

>PCR from Whole Cell Lysates


>1.  Pellet cells and resuspend in 1X PBS at a concentration of 2 x 10^6
cells/m*

>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
const*


>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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