Team:Cornell/Week 13
From 2011.igem.org
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:*'''Dephosphorylation of pSB1C3 5' Ends via CIAP Treatment''' | :*'''Dephosphorylation of pSB1C3 5' Ends via CIAP Treatment''' | ||
::- Please see the [https://2011.igem.org/Team:Cornell/Protocol Protocol] section for the procedure followed | ::- Please see the [https://2011.igem.org/Team:Cornell/Protocol Protocol] section for the procedure followed | ||
- | :*'''Gel Purification of Digestion Reaction | + | :*'''Gel Purification of Digestion Reaction Products via Electrophoresis''' |
- | ::- | + | ::- Please see the [https://2011.igem.org/Team:Cornell/Protocol Protocol] section for the procedure followed |
- | :*'''Gel Extraction | + | :*'''Gel Extraction of DNA''' |
- | ::- | + | ::- Please see the [https://2011.igem.org/Team:Cornell/Protocol Protocol] section for the procedure followed |
+ | ::- vioA and vioB genes migrated closely with their "native" pZE12 backbone, so initially both insert gene and backbone DNA bands were excised | ||
+ | ::- later decided to separate the two, but a labeling confusion prevented the exclusive purification and subsequent ligation of the insert gene (i.e., pZE12 backbone also underwent purification and ligation) | ||
:*'''NanoDrop Spectrophometry for Purified DNA Quantification''' | :*'''NanoDrop Spectrophometry for Purified DNA Quantification''' | ||
::- specific [ ]s to be posted soon | ::- specific [ ]s to be posted soon |
Revision as of 21:02, 11 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 28th - September 3rd
Sunday, August 28
Monday, August 29
Tuesday, August 30
Afternoon - Evening lab work done by: Claire Paduano, Youjin Cho, Maneesh Gupta, James Mathew, Charlie Chung
- Digestion Reactions for BioBrick Assembly
- Avi-Tagged RFP Insert
- 34.6µL ddH2O
- 7.9µL Avi-Tagged RFP DNA (1 µg)
- 5µL 10x NEBuffer 3
- 0.5µL 100x BSA
- 1µL EcoRI
- 1µL PstI
- 50µL Total
- Avi-Tagged vioA Insert
- 28.3µL ddH2O
- 14.2µL Avi-Tagged vioA DNA (1µg)
- 5µL 10x NEBuffer 3
- 0.5µL 100x BSA
- 1µL EcoRI
- 1µL PstI
- 50µL Total
- Avi-Tagged vioB Insert
- 26.7µL ddH2O
- 15.8µL Avi-Tagged vioB DNA (1µg)
- 5µL 10x NEBuffer 3
- 0.5µL 100x BSA
- 1µL EcoRI
- 1µL PstI
- 50µL Total
- Avi-Tagged vioE Insert
- 29.8µL ddH2O
- 11.7µL Avi-Tagged vioE DNA (1µg)
- 5µL 10x NEBuffer 3
- 0.5µL 100x BSA
- 1µL EcoRI
- 1µL PstI
- 50µL Total
- pSB1C3 Vector Backbone
- 40µL pSB1C3 DNA
- 5µL 10x NEBuffer 3
- 2.5µL ddH2O
- 0.5µL 100x BSA
- 1µL EcoRI
- 1µL PstI
- 50µL Total
- Dephosphorylation of pSB1C3 5' Ends via CIAP Treatment
- - Please see the Protocol section for the procedure followed
- Gel Purification of Digestion Reaction Products via Electrophoresis
- - Please see the Protocol section for the procedure followed
- Gel Extraction of DNA
- - Please see the Protocol section for the procedure followed
- - vioA and vioB genes migrated closely with their "native" pZE12 backbone, so initially both insert gene and backbone DNA bands were excised
- - later decided to separate the two, but a labeling confusion prevented the exclusive purification and subsequent ligation of the insert gene (i.e., pZE12 backbone also underwent purification and ligation)
- NanoDrop Spectrophometry for Purified DNA Quantification
- - specific [ ]s to be posted soon
- Ligation Reactions
- - specifics to be posted soon
Wednesday, August 31
Afternoon lab work done by: Youjin Cho
- Desalted the ligated samples from Tuesday and transformed them into MD37 electrocompetent cells using electroporation.
- After an hour of incubation, they were plated onto plate containing chloramphenicol.
Afternoon/Evening Lab work done by: Claire Paduano and Maneesh Gupta
- Looked at ATTO590 coated chip from Aug 27th under fluorescent microscope: channels still fluorescent
- Photo to be posted soon
- Streptavidin Coating and Fluorescent Probes
- 1) 45 min in 4% (by volume) MPTMS in ethanol
- 2) 20 min in 1mM GMBS
- 3) 45 min in 25ng/mL NeutrAvidin in PBS
- Streptavidin coating protocol from Gleghorn et al: http://www.ncbi.nlm.nih.gov/pubmed/20024046
- 4) 20 min in fluorescent probe
- Incubated one chip with ATTO520, one in ATTO590
- Flow Experiments
- Details and photos to be posted soon
- ATTO520 gives signal under both Texas Red and GFPA filters
- ATTO590 gives signal under only Texas Red
- -In future flow experimental design, keep in mind that we cannot differentiate between ATTO590 and 520 signal under Texas Red filter
Thursday, September 1
Morning lab work done by: Claire Paduano and Nancy Li
- Looked at ATTO590 coated chip from Aug 27th and chips from Aug31st under fluorescent microscope: channels still fluorescent
- Coated three chips with streptavidin
- Streptavidin coating
- Protocol from Gleghorn et al: http://www.ncbi.nlm.nih.gov/pubmed/20024046
- 1) 45 min in 4% (by volume) MPTMS in ethanol
- 2) 20 min in 1mM GMBS
- 3) 45 min in 25ng/mL NeutrAvidin in PBS
Evening lab work done by: Maneesh Gupta
- Did continuous flow experiments to test resilience of biotin-avitin binding
- Used same exposure (83.33) in all images taken
Friday, September 2
Afternoon lab work done by: Charlie Chung
- Miniprep DNA purification of cultures containing pSB1C3 vector backbone with (RFP+Avi-Tag), (vioA+Avi-Tag), (vioB+Avi-Tag), and (vioE+Avi-Tag) inserts -- in preparation for iGEM BioBrick submission
Saturday, September 3
Afternoon lab work done by: Jim Mathew, Charlie Chung, Nancy Li
- Quantification of Purified DNA Samples via NanoDrop Spectrophotometry
- - All 260/280nm ratios were near 1.8, indicting pure DNA
- - All inserts listed below are ligated with the Avi-Tagged pSB1C3 vector backbone
- RFP 1 -- 344.0ng/µL
- RFP 2 -- 256.4ng/µL
- RFP 3 -- 230.4ng/µL
- vioA 1-1 -- 205.9ng/µL
- vioA 1-2 -- 494.3ng/µL
- vioA 1-3 -- 370.8ng/µL
- vioA 2-1 -- 892.5ng/µL
- vioA 2-2 -- 203.8ng/µL
- vioA 2-3 -- 206.0ng/µL
- vioB 1-1 -- 503.9ng/µL
- vioB 1-2 -- 186.1ng/µL
- vioB 1-3 -- 398.8ng/µL
- vioB 2-1 -- 868.9ng/µL
- vioB 2-2 -- 145.6ng/µL
- vioB 2-3 -- 174.6ng/µL
- vioE 1 -- 266.2ng/µL
- vioE 2 -- 552.9ng/µL
- vioE 3 -- 400.7ng/µL
- DpnI Digestion of PCR Reaction Product
- - PCR with specially designed primers to delete the iGEM restriction enzyme cut sites, which may have been forming too much distance between the ribosome binding site and the start codon of our gene
- - first half of the forward primer consists of the base pairs flanking the deletion region to the left, second half of the forward primer consists of the base pairs flanking the deletion region to the right
- - when the primer anneals to the template, it is complementary to the nucleotides on either side of the deletion region and pinches them in, forcing the deletion region to loop out without a complementary strand
- Transformation via Electroporation
- Note: Either the DH5α or MC4100 strain will have the degradation system of unmethylated DNA knocked out
- - (RFP+Avi-Tag) into DH5α and MC4100
- - (vioA+Avi-Tag) into DH5α and MC4100
- - (vioB+Avi-Tag) into DH5α and MC4100
- - (vioE+Avi-Tag) into DH5α and MC4100
- Plating of Transformed Bacteria
- - Ampicillin-treated plates are incubating at 37°C in Weill Hall