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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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Verification digest to verify the presence of restriction sites in the psB1AC3, psB1AK3 vectors as well as in our TOPO TA vector with LuxOD47E.
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
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KATIE
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KEVIN
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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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