Team:Hawaii/Plasmid Prep

From 2008.igem.org

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=== Protocol===
=== Protocol===
1.  Grow single colony of ''E. coli'' at 37C overnight in 5 ml LB w/ antibiotic selection. <br>
1.  Grow single colony of ''E. coli'' at 37C overnight in 5 ml LB w/ antibiotic selection. <br>
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2.  Microcentrifuge 1.5 ml cells for 20 sec at 10,000g. Discard supernatant.<br>
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2.  Microcentrifuge 1.5 ml cells for 20 sec at 16,000g. Discard supernatant.<br>
3.  Resuspend pellet in 100 &mu;l GTE solution.<br>
3.  Resuspend pellet in 100 &mu;l GTE solution.<br>
:* 50 mM glucose
:* 50 mM glucose
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9.  Invert a few times to mix.<br>
9.  Invert a few times to mix.<br>
10. Incubate on ice for 5 min.<br>
10. Incubate on ice for 5 min.<br>
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11. Microcentrifuge for 3 min. at 10,000g.<br>
+
11. Microcentrifuge for 3 min. at 16,000g.<br>
12. Transfer supernatant to a new tube.<br>
12. Transfer supernatant to a new tube.<br>
13. Add 0.8 ml 95% ethanol.<br>
13. Add 0.8 ml 95% ethanol.<br>
14. Incubate for 2 min. at room temperature.<br>
14. Incubate for 2 min. at room temperature.<br>
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15. Microcentrifuge for 1 min. at 10,000g at room temperature. Remove supernatant.<br>
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15. Microcentrifuge for 1 min. at 16,000g at room temperature. Remove supernatant.<br>
16. Wash pellet w/ 1 ml 70% ethanol. Aspirate to dry (dry in hood).<br>
16. Wash pellet w/ 1 ml 70% ethanol. Aspirate to dry (dry in hood).<br>
17. Resuspend pellet in 30 &mu;l TE buffer.<br>
17. Resuspend pellet in 30 &mu;l TE buffer.<br>

Revision as of 04:49, 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

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

We prepped high copy plasmids:

  • pBluescript
  • pUC18
  • RP4

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

  • E0040
  • J04430
  • B0034
  • B0024

Result gel run:

Discusstion

Results

Discussion

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