"
Team:Cornell/Week 11
From 2011.igem.org
(Difference between revisions)
(→Monday, August 15) |
(→Monday, August 15) |
||
| Line 31: | Line 31: | ||
::*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: Maneesh Gupta, 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 #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) | |
| - | + | ::* 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 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 ???pm) | |
| - | + | ::* Incubate induced 25mL culture flask on room temperature shaker | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
==Tuesday, August 16== | ==Tuesday, August 16== | ||
Revision as of 22:42, 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 #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)
- 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 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 ???pm)
- Incubate induced 25mL culture flask on room temperature shaker
