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CAROL
QuikChange XL Site-Directed Mutagenesis for ''luxCDABE''in TOPO vector
Purpose: To mutate the XbaI site that is located about ~1000bp into the sequence. The reason for mutating the XbaI site for luxCDABE in TOPO vector is because to clone the sequence into a biobrick vector (pSB1AK3 and pSB1AC3), the enzyme, 'XbaI' is required for cloning purposes. Instead of cutting the sequence into two parts, we have to mutate the site so that it only cuts at the end of the sequence properly.
- QuikChange XL Site-Directed Mutagenesis Kit retrived from Stratagene
- Both the control reaction and the control transformation was done as well to ensure efficiency of mutagenesis. Please see detailed procedures for mutagenesis in the protocol section.
- Plamids were transformed into XL Gold Ultracompetent cells (part of the kit) and was plated on LB plates overnight at 37oC. The XL Gold Ultracompetent cells allow the uptake of single stranded DNA.
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CHINMOYEE
Research : What is Hill Function ?
Hill function was first used to describe the binding between hemoglobin and oxygen . The Hill function describe the binding affinities between enzymes and substrates.
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EMILY
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- Performed Antarctic Phosphotase treatment (NEB) on psB1AC3 vector from yesterday's construction digest according to the manufacturer's directions. Ligated with QuikLigase(Invitrogen).
- Transformed into TOP10 CaCl2 competant cells, along with PBluescript as a positive control for our competant cells, and plated on C plates for LuxOD47E and on A plates for PBluescript. Left plates in the 37 C incubator ovenright for growth.
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FAHD
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IMAN
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JAMIE
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JEREMY
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KATIE
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KEVIN
Bacterial transformation
The parts R0040, B0015, and J13002 were taken out from the 2009 DNA distribution kit, and were transformed into TOP10 cells. These parts are needed for general construction of our circuits.
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MANDY
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PATRICK
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PRIMA
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STEFAN
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VICKI
Plasmid isolation of LuxOD47A BBk from the overnight cultures made last night
Purpose: we have lots of competent TOP 10 cells with transformed LuxOD47A BBk on psB1AC3 in overnight plates, restreaks and cultures. The DNA in the overnight culture will be manipulated, but first it needs to be removed from the cells.
Protocol: Plasmid isolation was conducted in accordance with the procedure outlined on the protocol page for Sigma mini-prep kits. The concentrations of the isolated plasmids in ddHOH were measured with the Nanodrop spectrophotometer and placed in the -20 degree C freezer for a NotI restriction digest tomorrow.
Negative control test
Purpose: Contamination appears to be rampant in everyone’s PCR products. This will test our reagents used in PCR master mix and help determine where the contamination is coming from.
Protocol: 3 samples were made: the negative control PCR product from yesterday (already amplified); new master mix with old water; and new master mix with new water. A PCR with pTaq DNA polymerase was conducted in accordance with the procedure outlined on the protocol page.
Results: These are shown below. All of the purportedly negative controls are contaminated, including the new master mix made with new ddHOH. This implies that the communal reagents used in the master mix are also contaminated with a sequence that is specific to LuxO primers.
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