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CAROL
Isolation of Plasmids (pSB1AC3 and pSB1AK3)for Cloning
- Isolated plasmids (pSB1AK3 and pSB1AC3) from overnight cultures prepared by Thane.
- Used GenElute Plasmid Miniprep Kit (Sigma-Aldrich), used as according to the manufacturers instructions.
- Results:
| Plasmid | 260/280 | 260/230 | Concentration [ng/μL]
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| psB1AC3 1 | 1.96 | 3.48 | 76.9
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| psB1AC3 2 | 1.93 | 2.29 | 98.1
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| psB1AC3 3 | 1.87 | 1.88 | 48.5
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| psB1AC3 4 | 1.90 | 2.11 | 118.9
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| psB1AK3 1 | 2.05 | 2.42 | 134.4
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| psB1AK3 2 | 1.95 | 2.23 | 55.6
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| psB1AK3 3 | 1.91 | 2.22 | 97.9
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| psB1AK3 4 | 2.03 | 1.92 | 44.5
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- Set up restriction enzyme digest to ensure that biobrick sites are in the vector. The following are the reactions that were done:
Reaction 1: pSB1AC3 - cut with NotI
Reaction 2: pSB1AC3 - cut with EcoRI and SpeI
Reaction 3: pSB1AC3 - cut with XbaI and PstI
Reaction 4: pSB1AC3 - cut with XbaI and SpeI
Reaction 5: pSB1AK3 - cut with NotI
Reaction 6: pSB1AK3 - cut with EcorI and SpeI
Reaction 7: pSB1AK3 - cut with XbaI and PstI
Reaction 8: pSB1AK3 - cut with XbaI and SpeI
- Incubate reactions at 37oC
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CHINMOYEE
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EMILY
Purification of psB1AC3 & psB1AK3 vectors
- Protocol: Used GenElute Plasmid Miniprep Kit (Sigma-Aldrich), lot # 048k6062, used as according to the manufacturers instructions.
| Plasmid | 260/280 | 260/230 | Concentration [ng/μL]
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| psB1AC3 1 | 1.96 | 3.48 | 76.9
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| psB1AC3 2 | 1.93 | 2.29 | 98.1
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| psB1AC3 3 | 1.87 | 1.88 | 48.5
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| psB1AC3 4 | 1.90 | 2.11 | 118.9
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| psB1AK3 1 | 2.05 | 2.42 | 134.4
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| psB1AK3 2 | 1.95 | 2.23 | 55.6
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| psB1AK3 3 | 1.91 | 2.22 | 97.9
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| psB1AK3 4 | 2.03 | 1.92 | 44.5
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Restriction digest of psB1AC3 and psB1AK3 vectors as well as LuxOD47E in pCR.2.1-TOPO vector
- Objective: To verify the presence of restriction sites in the two BBK vectors as well as in our BBK gene in the pCR.2.1-TOPO vector
5 reactions for psB1AC3 vector, 5 reactions for psB1AK3 vector, 1 reaction for gene of interest (LuxOD47E in pCR2.1-TOPO vector).
BBK Vector Reaction List
Reaction 1- Not1 + React 3 Buffer
Reaction 2- EcoRI + SpeI + React Buffer 4
Reaction 3- XbaI + PstI + React 2 Buffer
Reaction 4- EcoRI + PstI + React 2 Buffer
Reaction 5- XbaI + SpeI + React 4 Buffer
BBK vector tube preparation
8 uL Plasmid (psB1AC3 or psB1AK3)
2 uL appropriate React Buffer
1 uL of each appropriate restrcition enzyme
ddH20 up to 20 uL
TOPO vector tube preparation
10 uL DNA (LuxOD47E in pCR2.1- TOPO vector)
7 uL ddH20
2 uL React 3 Buffer
1 uL NotI restriction enzymes
Left to digest overnight in waterbath at 37°C.
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FAHD
Descriptive Title of What You're Doing
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IMAN
Descriptive Title of What You're Doing
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JAMIE
Descriptive Title of What You're Doing
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JEREMY
Isolation and verification of pSB1AC3 and pSB1AK3
The iGEM lab team members worked together today to help eachother perfect the method of plasmid isolation. Plasmids psB1AC3 and psB1AK3 were isolated using Sigma GenElute Plasmid Mini-prep kit, and the concentration and purity of plasmid was measured using the NanoDrop Spectrophotometer. See Emily’s notebook for today (above) for results.
In order to verify that these vectors only had the death gene ccdB (675bp) in them, RD was set up with combinations of the following restriction enzymes: EcoRI, XbaI, PstI, NotI and SpeI. A gel was run with these products, as well as products from a RD set up with various genes of interest in their respective TOPO vectors (all cut with EcoRI).
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KATIE
Working with the Listen Event
Everything has to be added into chat so answers to questions are entered into the chat window, which I do not really like since mistakes are easy to make and I also keep communicating with a black board that uses one of the channels I am using as well.
Learned about the listen event:
- Uses channels and messages and keeps track of a avatar’s id so this will be very useful for: Giving out inventory, dividing messages up
Cylce of the PCR are complete now and an amplified piece of DNA, which is rezzed beside it and can be taken into the avatar’s inventory to be used for something else later on like running a gel.
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KEVIN
Plasmid Purification/isolation and verification of pSB1AC3
Plasmid Isolation
To isoloate pSB1AC3 plasmid from TOP10 cells, a miniprep on them was done using Sigma's GenElute plasmid Miniprep Kit (sigma). pSB1AC3 plasmids are needed for potential plasmid switches.
Table 1. Purity and concentration of pSB1AC3 measured with Nanodrop utility and Spectrophotometer
| Plasmid | 260/280 | 260/230 | Concentration [ng/μL]
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| pSB1AC3 C3 | 2.05 | 2.42 | 134.4
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| pSB1AC3 C4 | 1.95 | 2.23 | 55.6
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Restriction Digest
To verify if the biobrick restriction sites are present in pSB1AC3, restrition digest was done using all the different combinations of enzymes:
NotI, EcorI+SpeI, XbaI+PstI, EcorI+PstI and XbaI+SpeI
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MANDY
Descriptive Title of What You're Doing
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PATRICK
Descriptive Title of What You're Doing
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PRIMA
Biotech companies
Today I made an general email which I could sent to all Biotech companies. I had it checked by the rest of the marketing team. I had tailored some of the emails to target companies who had sponsored the building of the O'Brian labs and donated/offered quotes in the past. Since we had established a friendly long-term relationship with some of these companies, I wrote an email specifically to them regarding sponsoring this year's iGEM project.
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STEFAN
Descriptive Title of What You're Doing
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VICKI
Verification of vectors
Purpose: Check the BBk and TOPO vector tubes and confirm that they actually contain what the labels claim they do.
Materials and methods:
1. The plasmids (BBk and ccdB) came in E. coli. We performed a mini-prep to isolate them from the bacteria, in accordance with our plasmid isolation procedure. We were dealing with both psB1AC3 and psB1AK3 plasmids.
2. We evaluated the DNA concentrations with the Nanodrop spectrophotometer
3. We confirmed the contents with a restriction digest.
Restriction digest volumes
For TOPO plasmids: take 10 uL LuxPQ, 7 uL ddHOH
For restriction digest: 1 uL of each restriction enzyme, 2 uL of the relevant buffer, 8 uL of the plasmid and 9 uL of ddHOH – for a total volume of 20 uL
Reactions (enzymes that were used in cutting and relevant buffers):
- Tube 1 – NotI (REact 3)
- Tube 2 – EcoRI and SpeI (REact 4)
- Tube 3 – XbaI and PstI (REact 2)
- Tube 4 – EcoRI and PstI (REact 2)
- Tube 5 – XbaI and SpeI (REact 4)
- Tube 6 – TOPO with EcoRI (REact 3)
Results:
The restriction digest products were visualised on an agarose gel the following day.
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