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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, 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 Avi-Tag
- 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, gel purify, and ligate overnight
- PCR off GFP overnight
- 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 VioE in Avi-Tagged pZE12 vector backbone (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
- Ligation Reaction Setup
- vioB + Avi-Tagged pZE12 vector backbone
- 11.4μL pZE12 backbone (cut with KpnI & HindIII; former vioE gene digested out)
- 3.3μL vioA gene insert (cut with KpnI & HindIII)
- 2.3μL H2O
- 2μL 10x T4 DNA ligase buffer
- 1μL T4 DNA ligase
- 20μL Total
- Control Ligation for vioB Gene Inserts
- 11.4μL pZE12 backbone (cut with KpnI & HindIII; former vioE gene digested out)
- 5.6μL H2O (gene insert volumes go toward water volume)
- 2μL 10x T4 DNA ligase buffer
- 1μL T4 DNA ligase
- 20μL Total
- Incubate all ligation reaction constructs overnight in 16°C waterbath.
