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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
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IMAN
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JAMIE
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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
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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
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PATRICK
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PRIMA
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STEFAN
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VICKI
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