Daniel:Notebook/ComboLock/2016-8-23: Difference between revisions
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[[Category:ComboLock]] [[Category:20160822]] | [[Category:ComboLock]] [[Category:20160822]] [[Category:20160823]] |
Latest revision as of 22:42, 26 August 2016
Positive Control Amplicon Test 2 (Started yesterday)[edit]
Protocol[edit]
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 20 uL exonuclease I (20 units/μL) and 4 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- qPCR
- Set up reaction according to table
- Master Mix
- 277.5 uL KAPA SyberFast MM
- 194.25 uL nfH2O
- 5.55 uL AmpF 6.4
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
- 20160823-qPCRCurve-X2.png
X2 qPCR Curve
- 2016-08-23-PosConX2.png
X2 Gel Image
- 20160823-qPCRCurve-X3.png
X3 qPCR Curve
- 2016-08-23-PosConX3.png
X3 Gel Image
- 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)
- Add amplicons according to following table
- 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
Sample | Name | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 | X2-1X | A1 | 33 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | X2-5X | B1 | 34 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | X2-10X | C1 | 35 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | X2-50X | D1 | 36 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 | X3-1X | E1 | 37 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | X3-5X | F1 | 38 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 7 | X3-10X | G1 | 39 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 8 | X3-50X | H1 | 40 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9 | X2Flip | A12 | 41 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 10 | X3Flip | B12 | 42 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9 | PCR NTC | C12 | 43 | 2 | 25 | 0.5 | 5 | 19.5 | 50 |
Results[edit]
Positive Control Amplicon Production[edit]
Protocol[edit]
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 |
Sample | PCAmp1 | PCAmp3 | LatchX2 | LatchX3 |
AmpliconX1 | X | X | ||
AmpliconX3 | X | X |
Nanodrop Results[edit]
Sample | ng/uL ssDNA |
C1-C2 Amplicon | 287.6 |
C3-C2 Amplicon | 331.6 |
Protocol (Part 2)[edit]
File:2016-08-23-PosConAmplicon.png