Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2016/06/28: Difference between revisions

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:Heat inactivate rSAP at 75°C for 2 minutes
:Heat inactivate rSAP at 75°C for 2 minutes
:Heat inactivate BbsI at 65°C for 20 minutes
:Heat inactivate BbsI at 65°C for 20 minutes
*Digest pCer and pBta3R
:{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Volume (uL)'''
|-
| DNA (500ng)||12
|-
| 10x Green FD Buffer||3
|-
| EcoRI || 1
|-
| SpeI || 1
|-
| Water||13
|-
| Total||30
|}
:Incubate at 37°C for 10-20 minutes
:PCR purify


*PCR pExp and pLas  
*PCR pExp and pLas  

Revision as of 16:55, 1 July 2016

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06/28/2016

  • Digest LasR_MRV with BbsI
Reagent Volume (uL)
DNA (500ng) 1.5
10x Green FD Buffer 2.0
BbsI 1.0
Roche SAP 1.0
Water 14.5
Total 20
Incubate at 37°C for 10-20 minutes
Heat inactivate rSAP at 75°C for 2 minutes
Heat inactivate BbsI at 65°C for 20 minutes
  • Digest pCer and pBta3R
Reagent Volume (uL)
DNA (500ng) 12
10x Green FD Buffer 3
EcoRI 1
SpeI 1
Water 13
Total 30
Incubate at 37°C for 10-20 minutes
PCR purify


  • PCR pExp and pLas
Reagent Volume (uL)
F Primer 1
R Primer 1
DNA Template 1
Taq 2x MM 25
Water 22
Total 50
Thermocycler:
Temp (°C) Length (s)
95 30
95 15
60-70 30
72 15
72 5m
Extension for 15 minutes because of small size. Gradient of 60-70°C because pExp needs a higher annealing temperature to increase specificity; pLas uses 60°C to increase yield because it's plasmid is simple and does not contain similar binding sites. pExp was annealed at 66-70°C.


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