User:DrJones1935/10 August 2012
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(Created page with "====I. Digest pBAV1K-T5-gfp Plasmid and Cassette with XbaI and PstI==== *Mix (plasmid): :*37.5 uL water :*5 uL pBAV1K-T5-gfp DNA (~1 ug at 200 ng/uL) :*0.5 uL 100x BSA :*5 uL 10...") |
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=====''RESULTS''===== | =====''RESULTS''===== | ||
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''Concentration:'' | ''Concentration:'' |
Latest revision as of 01:40, 4 October 2012
Contents |
I. Digest pBAV1K-T5-gfp Plasmid and Cassette with XbaI and PstI
- Mix (plasmid):
- 37.5 uL water
- 5 uL pBAV1K-T5-gfp DNA (~1 ug at 200 ng/uL)
- 0.5 uL 100x BSA
- 5 uL 10 NEB Buffer 3
- 1 uL XbaI
- 1 uL PstI
- Mix (cassette):
- 10.32 uL water
- 32.18 uL cassette DNA (~1 ug at 31.075 ng/uL)
- 0.5 uL 100x BSA
- 5 uL 10 NEB Buffer 3
- 1 uL XbaI
- 1 uL PstI
- Incubate tubes in 37 oC water bath for 4 hours
II. QIAquick PCR Purification Protocol
- Add 5 volumes Buffer PB to 1 volume of reaction, mix
Reaction | Volume PB |
---|---|
plasmid - 50 uL | 250 uL |
cassette - 50 uL | 250 uL |
- Note: The solutions were all yellow, OK to proceed
- Apply samples to QIAquick columns, centrifuge for 1 minute at 13000 rpm, discard flow-through
- Add 750 uL of Buffer PE to the QIAquick column, centrifuge for 1 min and discard flow through
- Centrifuge again for 1 minute
- Transfer the column to clean microcentrifuge tube
- Elute cassette DNA by adding 30 uL of Buffer EB to the center of the column and let stand for 4 minutes
- Elute plasmid DNA by adding 30 uL of water to the center of the column and let stand for 4 minutes
- Centrifuge for 1 minute, store cassette DNA at 4 oC
III. Digest Restriction-digested Plasmid with CIP
- Mix:
- 28 uL of plasmid DNA (assume ~1 ug)
- 16 uL water
- 5 uL 10X NEB Buffer 3
- 1 uL diluted CIP
- 1 uL of CIP stock into 10 uL of water
- Incubate in 37 oC water bath for 1 hour
IV. QIAquick PCR Purification Protocol
- Add 5 volumes Buffer PB to 1 volume of reaction, mix
Reaction | Volume PB |
---|---|
plasmid - 50 uL | 250 uL |
- Note: The solutions were all yellow, OK to proceed
- Apply sample to QIAquick column, centrifuge for 1 minute at 13000 rpm, discard flow-through
- Add 750 uL of Buffer PE to the QIAquick column, centrifuge for 1 min and discard flow through
- Centrifuge again for 1 minute
- Transfer the column to clean microcentrifuge tube
- Elute olasmid DNA by adding 30 uL of Buffer EB to the center of the column and let stand for 4 minutes
- Centrifuge for 1 minute, store plasmid DNA at 4 oC
V. Measure the Concentration of Extracted DNA
- Bring samples and Buffer EB bottle upstairs to Take3 plate reader
- Add 2 uL of each sample according to the following chart:
Blank (EB) | Blank |
Blank | Blank |
plasmid | cassette |
- Use the program to blank the machine and measure sample concentrations
RESULTS
Concentration:
Sample | Concentration (ng/uL) | Approx. volume (uL) |
---|---|---|
plasmid | 19.714 | ~28 |
cassette | 15.692 | ~28 |