IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/20: Difference between revisions

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- Nanodrop
- Nanodrop
{|class="wikitable" style="text-align:center" border="1"
|-
! !! 260/280 !! ng/μL
|-
| AgrA || 1.66 || 16.4
|-
| AgrB || 1.91 || 23.5
|-
| AgrC || 1.99 || 35.9
|-
| AgrD || 2.13 || 4.9
|-
| Pxyl || 1.54 || 5.6
|-
| Ppac || 1.49 || 4.6
|-
| Pspc || 1.62 || 9.6
|-
| Pupp || 1.88 || 8.5
|-
| RBS S || 2.44 || 29
|-
| RBS W || 1.44 || 10.7
|-
|}


- Extract plasmid annd Agr from gel and clean
- Extract plasmid annd Agr from gel and clean

Revision as of 04:16, 21 August 2008

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Check Lux components

- Single colony PCR for :

  • R0040 (MIT stuff)
  • R0062 (MIT stuff)
  • 4 different colonies of S0168 (from a transformation plate from 12/08)
  • 4 different colonies of J04630 (from a transformation plate from 12/08)

- Protocol : add 1μL of cells (diluted in water), 10μL of Master Mis, 7μL of SDW, 1μL of VF primer and 1μL of VR primer

- Gel PCR products

- Results

  • R0040 and R0062 : one big band of about 300b (expected size 293), OK!
  • S0168 : one band of about 400b for the 4 different colonies (expected size 1234!), bad! This plate does not contain S0168
  • J04630 (colonies 2 and 4) : one band of about 1100b (expected size 1173), OK!
  • J04630 (colony 1) : one good band plus another band...
  • J04630 (colony 3) : one band of about 600b, bad!

Check vector ECE190

- Plasmid miniprep

- Nanodrop

- Double digest : 6μL of SDW, 8μL of DNA, 1μL of buffer, 1μL of XbaI and 1μL of PstI

- Run on a gel

- Result : 2 bands (a little bit more than 3000b and a little bit more than 5000b) :ok!

Ligation

- Materials :

  • AgrA
  • AgrB
  • AgrC
  • AgrD
  • Pupp
  • Pspac
  • Ppac
  • Pxyl
  • RBS S
  • RBS W
  • psB4C5

- Double digest of PCR products

- Run vector, AgrA and AgrD on a gel

- DNA clean and concentrator for AgrA, B,C and D, promoters

- Microclean for both RBS

- Nanodrop

260/280 ng/μL
AgrA 1.66 16.4
AgrB 1.91 23.5
AgrC 1.99 35.9
AgrD 2.13 4.9
Pxyl 1.54 5.6
Ppac 1.49 4.6
Pspc 1.62 9.6
Pupp 1.88 8.5
RBS S 2.44 29
RBS W 1.44 10.7

- Extract plasmid annd Agr from gel and clean

- Ligation


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