Team:Hawaii/Plasmid Prep

From 2008.igem.org

(Difference between revisions)
(→Discussion)
(→Discussion)
Line 44: Line 44:
== Discussion ==
== 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 A added to the NaOH/SDS solution.
+
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 A added.

Revision as of 21:07, 20 June 2008

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 tapping tube.
7. Incubate on ice for 5 min.
8. Add 150 μl potassium acetate solution.
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

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 A added.

Retrieved from "http://2008.igem.org/Team:Hawaii/Plasmid_Prep"