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Team:Cornell/Week 11
From 2011.igem.org
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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 (6:30pm start)
- At end of incubation time, check OD. Target OD = 0.6-0.8, which means ready for induction of RFP production via IPTG
- OD was 0.49 at 9pm. Let culture grow until 11pm for induction (IPTG addition).
- Induce 25mL RFP bacteria culture with 25µL 1M IPTG for desired 1mM addition (completed at 11pm)
- Incubate induced 25mL culture flask on room temperature shaker
Tuesday, August 16
Afternoon Lab Work Done By Maneesh Gupta and Charlie Chung
Repeated Sat. 13 Lysis protocol however after centrifuging the pellet was not red. Picked culture and incubated in shaker for miniprep and sequencing tomorrow.
