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Team:Cornell/Week 11
From 2011.igem.org
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Afternoon lab work done by: Maneesh Gupta, Charlie Chung | Afternoon lab work done by: Maneesh Gupta, Charlie Chung | ||
:*Repeated cell lysis protocol (see Saturday, August 13) on 25mL culture from Monday. | :*Repeated cell lysis protocol (see Saturday, August 13) on 25mL culture from Monday. | ||
| - | ::- However, after centrifuging, the pellet was not red | + | ::- However, after centrifuging, the pellet was not red |
:'''Troubleshooting Possibilities''' | :'''Troubleshooting Possibilities''' | ||
::*RFP being expressed in such little quantity that it cannot be confirmed by the naked eye | ::*RFP being expressed in such little quantity that it cannot be confirmed by the naked eye | ||
| - | :::- Instead, RFP expression can be confirmed with a western blot on the cell lysate using an antibody for | + | :::- Instead, RFP expression can be confirmed with a western blot on the cell lysate using an antibody for biotin |
::*Distance between the ribosomal binding site (RBS) and the AUG start codon of the RFP mRNA may be too long | ::*Distance between the ribosomal binding site (RBS) and the AUG start codon of the RFP mRNA may be too long | ||
:::- Redesign primers for the sake of cloning and expressing RFP | :::- Redesign primers for the sake of cloning and expressing RFP | ||
Revision as of 03:39, 16 September 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 + AviTagged pZE12 backbone) plates
- - GFP + AviTag + BB (1) #1, 2, 3
- - GFP + AviTag + BB (2) #1, 2, 3
- Incubating overnight in 37°C shaker of Room 304
- Re-picked three colonies from the (RFP + AviTagged pZE12 backbone) plate in order to retry subculturing, induction, and cell lysis
- - RFP + AviTag + 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 the Vio operon
- Miniprep & Sequencing
- Miniprepped the GFP samples using standard Qiagen Miniprep protocol
- GFP + AviTag + pZE12 backbone (1)- 1,2,3
- GFP + AviTag + 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 + AviTagged 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, Charlie Chung
- Repeated cell lysis protocol (see Saturday, August 13) on 25mL culture from Monday.
- - However, after centrifuging, the pellet was not red
- Troubleshooting Possibilities
- RFP being expressed in such little quantity that it cannot be confirmed by the naked eye
- - Instead, RFP expression can be confirmed with a western blot on the cell lysate using an antibody for biotin
- Distance between the ribosomal binding site (RBS) and the AUG start codon of the RFP mRNA may be too long
- - Redesign primers for the sake of cloning and expressing RFP
- What We Did
- - Picked a culture sample from the pellet formed after the centrifuge step
- - Incubated in the 37°C shaker of Room 304 for Miniprep and sequencing tomorrow
- p.s. -- Submit Didi's sequencing samples along with RFP sample!
Wednesday, August 17
Morning lab work done by: Youjin Cho, Charlie Chung
- Miniprep DNA Plasmid Purification
- Miniprepped culture from bacterial cell pellet of (RFP + Avi-Tagged pZE12)
- - Followed standard Qiagen Miniprep protocol
- - NanoDrop spectrophotometry: 52.7ng/μL
- Preparation for DNA Sequencing Submission
- - 1μL reverse primer for pZE12
- - 17μL Miniprep-purified (RFP + Avi-Tagged pZE12)
- Order Number: 10255141
Thursday, August 18
Friday, August 19
Lab work done by: Claire Paduano and Youjin Cho
- Objective
- PCR clean up VioB insert
- Digest VioB insert and VioE in Avi-Tagged pZE12 vector backbone and gel purification
- GFP sequencing came back: unsuccessful. PCR off GFP overnight to try ligation again
- Digestion Setups
- VioE in Avi-Tagged pZE12 vector backbone
- 19.05μL H2O
- 23.2μL VioE in Avi-Tagged pZE12 vector backbone (2μg)
- 5μL 10x NEBuffer 2
- 0.5μL 100x BSA
- 1.25μL KpnI
- 1μL HindIII
- 50μL Total
- VioB insert
- 38.25μL H2O
- 4μL VioB(1μg)
- 5μL 10x NEBuffer 2
- 0.5μL 100x BSA
- 1.25μL KpnI
- 1μL HindIII
- 50μL Total
- KpnI has only 75% efficiency in Buffer 2, so added 0.25uL more KpnI to digestion mixture
- Incubate in 37°C water bath for 2 hours
- Gel purification of VioB insert and Avi-Tagged pZE12 vector backbone
- VioB 5.4 ng/uL
- Avi-Tagged backbone 11.4 ng/uL
Saturday, August 20
Morning lab work done by: Claire Paduano
- Objective
- Set up ligation of VioB in Avi-Tagged backbone
- Ligation
- VioB + Avi-Tagged Backbone
- 2.3μL Avi-Tagged pZE12 vector backbone
- 14.7μL VioB
- 0.0μL H2O
- 2.0μL T4 DNA Ligase Buffer
- 1.0μL T4 DNA Ligase
- 20μL Total
- In all above reactions, volume of plasmid vector added corresponds with 100ng backbone.
- Prepare control ligation reactions (inserts replaced with H2O) for each of the above constructs.
- Same volume of vector backbone, buffer, ligase
- Volumes of all inserts (i.e. gene) go toward volume of H2O
- After ligation setup, incubate at room temperature for 4hrs.
Afternoon lab work done by: Youjin Cho, Charlie Chung
- vioB Transformation
- Desalted the (vioB + Avi-Tag + BB) ligation reaction product and the (Avi-Tagged BB) control
- Transformed (vioB + Avi-Tag + BB) and the (Avi-Tagged BB) control into DH5α bacteria via electroporation
- Plated onto (LB + ampicillin) dishes and incubated overnight at 37°C
- Alternative Method for RFP Expression
- Sequencing results from Wednesday's (August 17) sample show that the RFP gene was present in the Avi-Tagged pZE12 vector backbone
- Transformed 1.5μL of (RFP + Avi-Tag + BB) Miniprep-purified plasmid into DH5α bacteria with the IPTG gene via electroporation
- - Ideal Results: Colonies that have grown tomorrow will be red because RFP-production is constitutively on
- Plated onto a (LB + ampicillin) dish and incubated overnight at 37°C
