Daniel:Notebook/ComboLock/2016-9-28: Difference between revisions
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| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 557.1 | ||
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Revision as of 22:06, 28 September 2016
Positive Control Amplicon Production (PCAmp4)
Protocol
- Phosphorylation
- In a 0.2 mL tube, add ingredients according to table
- Incubate at 37C for 30 min
- Ligation
- Set up 2 reactions with the following reagents (DO NOT ADD LIGASE YET)
- Heat reaction to 95C for 5 min
- Lower temp to 55C
- Add 1 uL Amp Ligase to each reaction without removing from thermocycler; swirl with pipette tip 5 times to mix
- Incubate at 55C for 2 hours
- Heat to 95 C to denature dsDNA
- Purify with ssDNA column
- ssDNA Column
- Add 100 uL Binding Buffer to the sample; mix well
- Transfer to IIC Column and centrifuge at 14000 rpm for 1 minute; SAVE THE FLOW THROUGH
- Add 150 uL 100% EtOH to flow through; mix well
- Transfer to IC Column and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 400 uL Prep Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 700 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Add 400 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
- Centrifuge empty column at 14000 rpm for 2 minutes
- Transfer to empty 1.5mL centrifuge tube (low bind)
- Add 30 uL nfH2O and centrifuge at 14000 rpm for 1 minute
- TBE Gel
- Mix 32 uL TBE and 8 uL 6x Gel Loading dye
- Aliquot 10 uL for each sample to parafilm
- Add 2 uL of diluted sample/ladder to the drops
- Mix with the loading pipette and add 10 uL to each lane
- Run for 25 min at 250V
- Stain with 3 uL SYBR Gold for 3 min
- Rinse and image (see gallery below)
- Size Select
- For sample lanes, mix 16 uL sample, 14 uL 6X loading dye and 50 uL TBE (80 uL total)
- For ladder lanes, mix 4 uL 25bp ladder, 6 uL 6X loading dye and 30 uL TBE (40 uL total)
- Add 20 uL to each lane (2 gels total, 1 per sample)
- Run gel for 25 minutes at 250V
- While running, prepare tubes by puncturing a 0.5 mL tube with a 0.22 gauge needle and putting inside a 1.5 mL tube
- Stain gel for 3 minutes with 3 uL SYBR gold
- Image in gel doc
- Extract bands at 104 bp and put into tubes; 2 lanes per tube (4 tubes total, 2 per sample)
- Image in gel doc post-extraction
- Centrifuge tubes at 12000rpm for 1.5 minutes
- Discard 0.5 mL tube and resuspend in 500 uL TE buffer
- Incubate at 37C for 1 hour with vigorous shaker
- Centrifuge at 12000 rpm for 1.5 minutes
- Extract the supernatant and place into NanoSep column
- Centrifuge at 12000 rpm for 1.5 minutes
- Ethanol Precipitation
- Move sample to a fresh 2 mL tube
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc and 2 uL glycoblue
- Store at -80C overnight, continued tomorrow
Reagent | Stock Conc | Final Conc./Amount | uL added |
AmpLigase Reaction Buffer | 10X | 1X | 2 |
ATP | 10 mM | 1 mM | 2 |
PCAmp2 | 100 uM | 1 nmol total | 10 |
T4 DNA Kinase | 10 U/uL | 10 U | 1 |
nfH2O | NA | NA | 5 |
Total | 20 |
Reagent | Stock Conc | Final Conc./Amount | uL added |
AmpLigase Reaction Buffer | 10X | 1X | 4 |
Latch Oligo | 100 uM | 500 umol | 5 |
5' Amplicon Oligo | 100 uM | 500 umol | 5 |
Amp Ligase | 5 U/uL | 5 U | 1 |
Phosphate Reaction | NA | NA | 10 |
nfH2O | NA | NA | 25 |
Total | 50 |
Nanodrop Results
Sample | ng/uL ssDNA |
C4-C2 Amplicon | 557.1 |
Protocol (Part 2)
[[Image:|500px]]