Team:Cambridge/LabBook/Week6

From 2010.igem.org

(Difference between revisions)
(New page: ===42. Experiment: Transforming TOP10 with pBAD promoter (Hannah)=== Took plasmid from Shuna, followed protocol from earlier, growing up stock to make more plasmid so we can PCR from it la...)
Line 22: Line 22:
|
|
| 89.2
| 89.2
 +
|}
 +
 +
These are in storage in the -20°C
 +
 +
===44. Experiment: Growth of Slock10, TOP10 pHK555+pHK724 and phosphoreum to make glycerol stocks for tomorrow (Hannah)===
 +
*Used antibiotic resistance markers to select for bacteria in Slock10 + TOP10
 +
*These were put in the 37°C incubator
 +
 +
===45. Experiment: Growing up of 10ml overnight cultures of old glowth (from non-glowing flask) and BBa_216007 (Peter)===
 +
Made 4 vials:
 +
*Amp + Chl      glowth?
 +
*Amp            glowth?
 +
*Amp            glowth? + lumazine (BBa_216007), double inoculation
 +
*Amp            lumazine
 +
 +
Placed in incubator at 30°C
 +
 +
===46. Experiment: Transformation of TOP10cc with BBa_R0040 (TetR repressed promoter), BBa_E0030 (enhanced yellow fluorescent protein) separately. (Theo & Anja)===
 +
BBa_R0040 (Plate 1, Well 6L) and BBa_E0030 (Plate 2, Well 24E) were resuspended in 10μl deionised H20.
 +
 +
TOP10cc were taken out of -80°C freezer and thawed on ice. 1ml pipette tip cut with scissors (sterile). 2x50μl of TOP10cc were transformed to 1.5ml eppendorf tubes.
 +
 +
2μl of resuspended BBa_R0040 and 2μl of resuspended BBa_E0030 were added to separate eppendorf tubes with TOP10cc. Cells were held on ice for 30mins. Heat shocked for 60s at 42°C (waterbath). Put on ice for ~2mins. Added 250μl pre-warmed (37°C) soc. Incubated at 37°C for 1h with rotation. Plated 50μl on pre-warmed (in 37°C incubator) LB agar plates with:
 +
*Amp for BBa_R0040
 +
*Kan for BBa_E0030
 +
 +
Grow colonies overnight at 37°C.
 +
 +
===46. Experiment: Streaking out and setting up overnight cultures of TOP10 with promoter + rbs + luc (Anja)===
 +
Streaked out from a single colony on an LB agar plate with Amp and incubate at 37°C overnight.
 +
 +
Inoculated single bacterial colony in 5ml LB with 5μl (100mg/ml) Ampicillin, incubated at 37°C with shaking overnight.
 +
 +
===47. Experiment: Using plate reader to characterise light output of the pHK724+pHK555 in Slock10 and TOP10 as they grew. (Paul)===
 +
Grew the following (1/2 Slock10, 1/2 TOP10):
 +
*10 x Cells + Medium LB + Amp
 +
*10 x Cells + Medium LB + Chl
 +
*10 x Cells + Medium LB + Amp + Chl
 +
 +
Final concentrations of Amp = 100μg/ml, Chl = 20μg/ml
 +
 +
Growth - at 30°C as we hope this will maximise light output.
 +
 +
Volume per well - 100μl of total (= LB + Chl/Amp + Cells)
 +
 +
Cells used - grown up in broth on previous day by Paul in LB + antibiotic broth.
 +
 +
===48. Experiment: Making long-term stock of TOP10 with promoter + rbs + p.p.luc (Peter)===
 +
Added 0.5ml 40% glycerol solution and 0.5ml sample culture to cryogenic vial (x2). Gently vortexed.
 +
 +
Stored in -80°C freezer in 'Black-GM230hns-205' storage box. Vials labelled with sticker 'TetR luc'.
 +
 +
===49. Experiment: Transfer of promoter + rbs + p.p.luc into psB1C3 for submission to registry. (Will, Peter & Anja)===
 +
1.5ml of TOP10 cells with promoter+rbs+luc grown overnight in LB+Amp were transferred to eppendorf tubes (x2) and cells were spun down in microcentrifuge (1min, 13000rpm). Suernatant was discarded and plasmid was purified following the 'QIAprep Spin Miniprep Kit using a microcentrifuge' protocol.
 +
 +
Nanodrops showed 135.1ng/μl DNA present in purified plasmid prep (EB as blank).
 +
 +
====Restriction enzyme digest====
 +
Prepared at room temperature in the listed order:
 +
{| class="wikitable"
 +
|-
 +
| nuclease-free H20
 +
| 14μl
 +
|-
 +
| 10x Fast Digest Buffer
 +
| 2μl
 +
|-
 +
| Plasmid DNA
 +
| 2μl
 +
|-
 +
| FD Enzyme EcoRI
 +
| 1μl
 +
|-
 +
| FD Enzyme PstI
 +
| 1μl
 +
|-
 +
|
 +
| 20μl
|}
|}

Revision as of 09:35, 26 August 2010

Contents

42. Experiment: Transforming TOP10 with pBAD promoter (Hannah)

Took plasmid from Shuna, followed protocol from earlier, growing up stock to make more plasmid so we can PCR from it later.

Plated out on Kanomycin plates

43. Experiment: Plasmid extraction - pHK555 + LuxR from registry (Hannah)

Followed Qiagen miniprep protocol

Plasmid Concentration (ng/μl)
pHK555 20.9
17.3
LuxR 43.9
89.2

These are in storage in the -20°C

44. Experiment: Growth of Slock10, TOP10 pHK555+pHK724 and phosphoreum to make glycerol stocks for tomorrow (Hannah)

  • Used antibiotic resistance markers to select for bacteria in Slock10 + TOP10
  • These were put in the 37°C incubator

45. Experiment: Growing up of 10ml overnight cultures of old glowth (from non-glowing flask) and BBa_216007 (Peter)

Made 4 vials:

  • Amp + Chl glowth?
  • Amp glowth?
  • Amp glowth? + lumazine (BBa_216007), double inoculation
  • Amp lumazine

Placed in incubator at 30°C

46. Experiment: Transformation of TOP10cc with BBa_R0040 (TetR repressed promoter), BBa_E0030 (enhanced yellow fluorescent protein) separately. (Theo & Anja)

BBa_R0040 (Plate 1, Well 6L) and BBa_E0030 (Plate 2, Well 24E) were resuspended in 10μl deionised H20.

TOP10cc were taken out of -80°C freezer and thawed on ice. 1ml pipette tip cut with scissors (sterile). 2x50μl of TOP10cc were transformed to 1.5ml eppendorf tubes.

2μl of resuspended BBa_R0040 and 2μl of resuspended BBa_E0030 were added to separate eppendorf tubes with TOP10cc. Cells were held on ice for 30mins. Heat shocked for 60s at 42°C (waterbath). Put on ice for ~2mins. Added 250μl pre-warmed (37°C) soc. Incubated at 37°C for 1h with rotation. Plated 50μl on pre-warmed (in 37°C incubator) LB agar plates with:

  • Amp for BBa_R0040
  • Kan for BBa_E0030

Grow colonies overnight at 37°C.

46. Experiment: Streaking out and setting up overnight cultures of TOP10 with promoter + rbs + luc (Anja)

Streaked out from a single colony on an LB agar plate with Amp and incubate at 37°C overnight.

Inoculated single bacterial colony in 5ml LB with 5μl (100mg/ml) Ampicillin, incubated at 37°C with shaking overnight.

47. Experiment: Using plate reader to characterise light output of the pHK724+pHK555 in Slock10 and TOP10 as they grew. (Paul)

Grew the following (1/2 Slock10, 1/2 TOP10):

  • 10 x Cells + Medium LB + Amp
  • 10 x Cells + Medium LB + Chl
  • 10 x Cells + Medium LB + Amp + Chl

Final concentrations of Amp = 100μg/ml, Chl = 20μg/ml

Growth - at 30°C as we hope this will maximise light output.

Volume per well - 100μl of total (= LB + Chl/Amp + Cells)

Cells used - grown up in broth on previous day by Paul in LB + antibiotic broth.

48. Experiment: Making long-term stock of TOP10 with promoter + rbs + p.p.luc (Peter)

Added 0.5ml 40% glycerol solution and 0.5ml sample culture to cryogenic vial (x2). Gently vortexed.

Stored in -80°C freezer in 'Black-GM230hns-205' storage box. Vials labelled with sticker 'TetR luc'.

49. Experiment: Transfer of promoter + rbs + p.p.luc into psB1C3 for submission to registry. (Will, Peter & Anja)

1.5ml of TOP10 cells with promoter+rbs+luc grown overnight in LB+Amp were transferred to eppendorf tubes (x2) and cells were spun down in microcentrifuge (1min, 13000rpm). Suernatant was discarded and plasmid was purified following the 'QIAprep Spin Miniprep Kit using a microcentrifuge' protocol.

Nanodrops showed 135.1ng/μl DNA present in purified plasmid prep (EB as blank).

Restriction enzyme digest

Prepared at room temperature in the listed order:

nuclease-free H20 14μl
10x Fast Digest Buffer 2μl
Plasmid DNA 2μl
FD Enzyme EcoRI 1μl
FD Enzyme PstI 1μl
20μl
Retrieved from "http://2010.igem.org/Team:Cambridge/LabBook/Week6"