Team:Cornell/Week 11

From 2011.igem.org

(Difference between revisions)
(Monday, August 15)
(Monday, August 15)
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Evening lab work done by: Maneesh Gupta, Charlie Chung
Evening lab work done by: Maneesh Gupta, Charlie Chung
:'''Preparing a Subculture'''
:'''Preparing a Subculture'''
-
::* Set up two cuvettes for preliminary optical density (OD) reading: (1) control (2) RFP sample from Thursday evening  
+
::* Set up two cuvettes for preliminary optical density (OD) reading: (1) control (2) RFP sample from Sunday evening  
:::- Purpose of preliminary OD reading is to determine how much RFP sample you need to add to a new 25mL culture  
:::- Purpose of preliminary OD reading is to determine how much RFP sample you need to add to a new 25mL culture  
:::- Use a 1:10 dilution of sample to minimize error when running the spectrophotometer
:::- Use a 1:10 dilution of sample to minimize error when running the spectrophotometer
::::* Control: 1000µL LB
::::* Control: 1000µL LB
::::* RFP Sample: 900µL LB + 100µL (RFP + Avi-Tagged pZE12) Colony #9  
::::* RFP Sample: 900µL LB + 100µL (RFP + Avi-Tagged pZE12) Colony #9  
-
::* RFP Sample OD = 0.329 (treat as [bacteria with RFP]), which translates to actual OD of 3.29 in Thursday's 5mL culture tube (after undoing the 1:10 dilution)  
+
::* RFP Sample OD = 0.329 (treat as [bacteria with RFP]), which translates to actual OD of 3.29 in Sunday's 3mL culture tube (after undoing the 1:10 dilution)  
::* Use dilution equation to determine how much RFP bacteria culture is needed for the 25mL culture  
::* Use dilution equation to determine how much RFP bacteria culture is needed for the 25mL culture  
-
:::(3.29)(? µL) = (desired beginning [RFP bacteria] = 0.05)(25mL = 25000µL)  
+
::::(3.29)(? µL) = (desired beginning [RFP bacteria] = 0.05)(25mL = 25000µL)  
-
:::? = 380µL Thursday's RFP bacteria culture to 25mL LB + 25µL ampicillin  
+
::::? = 380µL Sunday's RFP bacteria culture to 25mL LB + 25µL ampicillin  
::* Incubate new 25mL RFP bacteria subculture in 37°C shaker for ~2 hours and 45 minutes
::* Incubate new 25mL RFP bacteria subculture in 37°C shaker for ~2 hours and 45 minutes
::* At end of incubation time, check OD. Target OD = 0.6-0.8, which means ready for induction of RFP production via IPTG
::* At end of incubation time, check OD. Target OD = 0.6-0.8, which means ready for induction of RFP production via IPTG

Revision as of 22:43, 15 August 2011

Results | Protocol | Notebook | Parts Submitted

Week 1 | Week 2 - 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18 | Week 19 | Week 20 | Week 21 |


August 14th - August 20th

Sunday, August 14

Afternoon lab work done by: Charlie Chung

  • Picked three colonies from the two (GFP + Avi-Tagged pZE12 backbone) plates
- GFP + Avi + BB (1) #1, 2, 3
- GFP + Avi + BB (2) #1, 2, 3
  • Incubating overnight in 37°C shaker of Room 304
  • Re-picked three colonies from the (RFP + Avi-Tagged pZE12 backbone) plate in order to retry subculturing, induction, and cell lysis
- RFP + Avi + BB #7, 8, 9
  • Incubating overnight in 37°C shaker of Room 304

Monday, August 15

Afternoon lab work done by: Youjin Cho, Maneesh Gupta

Objective
  • Miniprep GFP samples that were cultured overnight and send them off for sequencing.
  • Set-up the PCR for vioB using vio operon.
Miniprep & Sequencing
  • Miniprepped the GFP samples using standard Qiagen Miniprep protocol.
GFP+Avi-Tag+pZE12 backbone (1)- 1,2,3
GFP+Avi-Tag+pZE12 backbone (2)- 1,2,3
  • Set-up the samples for sequencing using the reverse primer.
Order number: 10254977
PCR Reaction
  • Set-up the vioB PCR using the vio operon (= 20.3ng/μL).
  • As Didi suggested, used the melting temperature of 53°C and annealing time for 3:30 minutes.

Evening lab work done by: Maneesh Gupta, Charlie Chung

Preparing a Subculture
  • Set up two cuvettes for preliminary optical density (OD) reading: (1) control (2) RFP sample from Sunday evening
- Purpose of preliminary OD reading is to determine how much RFP sample you need to add to a new 25mL culture
- Use a 1:10 dilution of sample to minimize error when running the spectrophotometer
  • Control: 1000µL LB
  • RFP Sample: 900µL LB + 100µL (RFP + Avi-Tagged pZE12) Colony #9
  • RFP Sample OD = 0.329 (treat as [bacteria with RFP]), which translates to actual OD of 3.29 in Sunday's 3mL culture tube (after undoing the 1:10 dilution)
  • Use dilution equation to determine how much RFP bacteria culture is needed for the 25mL culture
(3.29)(? µL) = (desired beginning [RFP bacteria] = 0.05)(25mL = 25000µL)
? = 380µL Sunday's RFP bacteria culture to 25mL LB + 25µL ampicillin
  • Incubate new 25mL RFP bacteria subculture in 37°C shaker for ~2 hours and 45 minutes
  • At end of incubation time, check OD. Target OD = 0.6-0.8, which means ready for induction of RFP production via IPTG
  • Induce 25mL RFP bacteria culture with 25µL 1M IPTG for desired 1mM addition (completed at ???pm)
  • Incubate induced 25mL culture flask on room temperature shaker

Tuesday, August 16

Wednesday, August 17

Thursday, August 18

Friday, August 19

Saturday, August 20