Team:Cambridge/Bacillus/Lab Work

From 2008.igem.org

(Difference between revisions)
(→July 22nd)
(→July 22nd)
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*Take 15uL of each and put it on plates without antibiotic
*Take 15uL of each and put it on plates without antibiotic
*Incubate at 37°C
*Incubate at 37°C
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 +
 +
=July 23rd=
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* Take one colony from each of the plates grown from the tubes yesterday and plated them again on LA without antibiotics
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 +
 +
 +
'''1/ Purpose''' Check antibiotic resistance of our different strains
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 +
{|class="wikitable" style="text-align:center" border="1"
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|-
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! Strain
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! Plasmid
 +
! Extracted from
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! Antibiotic use
 +
! Concentration of antibiotic
 +
|-
 +
| DH5alpha || PP182 || tube || Amp || 100
 +
|-
 +
| MC1061 || NONE || tube || Cm || 35
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|-
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| MC1061 || PNZ8901 || tube || Cm || 35
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|-
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| DH5alpha || PP182 || plate || Amp || 100
 +
|-
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| MC1061 || NONE || plate || Cm || 35
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|-
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| MC1061 || PNZ8901 || plate || Cm || 35
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|-
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|}
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 +
 +
'''2/ Purpose''' : Find the right concentration of antibiotic so that B. subtilis survive
 +
 +
- Grow 15uL of B. 1A1 (frozen tube) in 5mL of LB
 +
 +
- Incubate at 37°C
 +
 +
'''3/ Purpose ''': Grow plasmids in TOP10, transformation
 +
 +
4 plasmids :
 +
* I746000
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* I746100
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* I746101
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* I746001
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 +
1 control : PUC19
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 +
- Add 20uL of TOP10 competent cells and 0.5uL of Plasmid in an eppendorf
 +
 +
- 30min on ice
 +
 +
- Heat shock : 60s at 42°C
 +
 +
- 2min on ice
 +
 +
- Add 89.5uL of SOC
 +
 +
- 60min at 37°C
 +
 +
- Put the mix on plate with ampicillin resistance
 +
 +
- Incubate at 37°C

Revision as of 17:52, 28 October 2008

July 22nd

We have 4 tubes from last year. These strains are frozen in glycerol.

  • PNZ8901 plasmid in E.coli MC1061 Strain
    • Chloroamphenicol resistant
    • "sure" vector
    • does not integrate
  • B. subtilis strain 1A1
    • No resistance
    • Same as strain 168 but tryptophan deficient
  • E.coli strain MC1061, no plasmid
  • PP182 plasmid in E.coli strain DH5alpha
    • Ampicillin resistance
    • integrates


Checking antibiotic resistance

Purpose : Grow each of them on a plate to test their antibiotic resistance

Protocol

  • Warm up frozen tubes
  • Take 15uL of each and put it on plates without antibiotic
  • Incubate at 37°C


July 23rd

  • Take one colony from each of the plates grown from the tubes yesterday and plated them again on LA without antibiotics


1/ Purpose Check antibiotic resistance of our different strains

Strain Plasmid Extracted from Antibiotic use Concentration of antibiotic
DH5alpha PP182 tube Amp 100
MC1061 NONE tube Cm 35
MC1061 PNZ8901 tube Cm 35
DH5alpha PP182 plate Amp 100
MC1061 NONE plate Cm 35
MC1061 PNZ8901 plate Cm 35


2/ Purpose : Find the right concentration of antibiotic so that B. subtilis survive

- Grow 15uL of B. 1A1 (frozen tube) in 5mL of LB

- Incubate at 37°C

3/ Purpose : Grow plasmids in TOP10, transformation

4 plasmids :

  • I746000
  • I746100
  • I746101
  • I746001

1 control : PUC19

- Add 20uL of TOP10 competent cells and 0.5uL of Plasmid in an eppendorf

- 30min on ice

- Heat shock : 60s at 42°C

- 2min on ice

- Add 89.5uL of SOC

- 60min at 37°C

- Put the mix on plate with ampicillin resistance

- Incubate at 37°C





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