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Team:Cornell/Week 11
From 2011.igem.org
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::*Set-up the vioB PCR using the vio operon (= 20.3ng/μL). | ::*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. | ::*As Didi suggested, used the melting temperature of 53°C and annealing time for 3:30 minutes. | ||
| - | Evening lab work done by: Charlie Chung | + | 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 Thursday 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 #5 | ||
| + | |||
| + | * RFP Sample OD = 0.283 (treat as [bacteria with RFP]), which translates to actual OD of 2.83 in Thursday's 5mL culture tube (after undoing the 1:10 dilution) | ||
| + | |||
| + | * Use dilution equation to determine how much RFP bacteria culture is needed for the 25mL culture | ||
| + | |||
| + | (2.83)(? µL) = (desired beginning [RFP bacteria] = 0.05)(25mL = 25000µL) | ||
| + | ? = 441.7µL Thursday'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 7:25pm) | ||
| + | * Incubate induced 25mL culture flask on room temperature shaker | ||
==Tuesday, August 16== | ==Tuesday, August 16== | ||
Revision as of 22:34, 15 August 2011
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 Thursday 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 #5
* RFP Sample OD = 0.283 (treat as [bacteria with RFP]), which translates to actual OD of 2.83 in Thursday's 5mL culture tube (after undoing the 1:10 dilution)
* Use dilution equation to determine how much RFP bacteria culture is needed for the 25mL culture
(2.83)(? µL) = (desired beginning [RFP bacteria] = 0.05)(25mL = 25000µL)
? = 441.7µL Thursday'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 7:25pm)
* Incubate induced 25mL culture flask on room temperature shaker
