Team:Hawaii/Plasmid Prep

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Revision as of 03:57, 8 July 2008 by Gracek (Talk | contribs)

Contents

Protocol

1. Grow single colony of E. coli at 37C overnight in 5 ml LB w/ antibiotic selection.
2. Microcentrifuge 1.5 ml cells for 20 sec at 16,000g. Discard supernatant.
3. Resuspend pellet in 100 μl GTE solution.

  • 50 mM glucose
  • 10 mM EDTA
  • 25 mM Tris-HCl (pH 8.0)

4. Let sit for 5 min. at room temperature.
5. Add 200 μl NaOH/SDS solution.

  • 0.2 M NaOH
  • 1% SDS

6. Mix by inverting the tube a few times.
7. Incubate on ice for 5 min.

  • Incubate no more than 5 min. to allow for maximum release of plasmid DNA while minimizing genomic DNA release and overexposure to denaturing conditions.

8. Add 150 μl potassium acetate solution.

  • Precipitation of cellular debris may be enhanced by using chilled KOAc.

9. Invert a few times to mix.
10. Incubate on ice for 5 min.
11. Microcentrifuge for 3 min. at 16,000g.
12. Transfer supernatant to a new tube.
13. Add 0.8 ml 95% ethanol.
14. Incubate for 2 min. at room temperature.
15. Microcentrifuge for 1 min. at 16,000g at room temperature. Remove supernatant.
16. Wash pellet w/ 1 ml 70% ethanol. Aspirate to dry (dry in hood).
17. Resuspend pellet in 30 μl TE buffer.

Reference: Short Protocols in Molecular Biology

  • Add 1 μl RNase after step 12. Incubate at 55C for 30-90 min. (Re: SC)

Results

First attempt on 2008.06.17

Failed, we should see something bigger than a smear between 100kb-500kb, this run uses 10ul plasmid-prep, 2ul loading dye

According to Gernot, we may have sheared the plasmid DNA (he has not seen the picture). Another problem may be that pRL1383a is a LOW copy plasmid. we should start with more material instead of the standard 1.5mL spindown per 30ul of TE suspended final plasmid.

Second attempt on 2008.06.19

What is that stuff between 100bp and 500bp? this run is half as bright because i'm using 5ul plasmid-prep, 2ul loading dye

We prepped biobrick plasmids (high copy?) (assuming these clones have the parts we want on them):

  • E0040
  • J04430
  • B0034
  • B0024

Regular Plasmids

  • pBluescript
  • pUC18
  • RP1
  • pBluescript (vortexed, pippetted up down few times when loading, to see if it's the cause of the small fragments.)

Discussion

Plasmid was probably sheared the first time. Band at 100-500bp is likely RNA, rRNA to be specific. To confirm that the band is RNA, we should digest the band with RNase and run it through the gel again. Alternatively, we can try another plasmid prep with RNase added. The Qiagen plasmid prep kit adds RNase in the NaOH/SDS solution.

GP suggests we add RNase as early as possible. It will be added alongside GTE.

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