Daniel:Notebook/ComboLock/2016-9-28: Difference between revisions
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>Djacobse (Created page with "=Positive Control Amplicon Production (PCAmp4)= Back to Calendar ==Protocol== <ol> <li>Phosphorylation</li> <ol type="A"> <li>In a 0.2 mL tube...") |
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| height="15" valign="bottom" | | | height="15" valign="bottom" | C4-C2 Amplicon | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 557.1 | ||
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==Protocol (Part 2)== | |||
<ol start="4"> | |||
<li>Size Select</li> | |||
<ol type="A"> | |||
<li>For sample lanes, mix 32 uL sample, 28 uL 6X loading dye and 100 uL TBE (160 uL total)</li> | |||
<li>For ladder lanes, mix 4 uL 25bp ladder, 6 uL 6X loading dye and 30 uL TBE (40 uL total)</li> | |||
<li>Add 20 uL to each lane (2 gels total, 1 per sample)</li> | |||
<li>Run gel for 25 minutes at 250V</li> | |||
<li>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</li> | |||
<li>Stain gel for 3 minutes with 3 uL SYBR gold</li> | |||
<li>Image in gel doc</li> | |||
<li>Extract bands at 104 bp and put into tubes; 2 lanes per tube (4 tubes total, 2 per sample)</li> | |||
<li>Image in gel doc post-extraction</li> | |||
<gallery perrow=2 heights=300px widths=300px> | |||
File:2016-09-28-PCAmp4Prod-SizeSelect.png|Before Image | |||
File:2016-09-28-PCAmp4Prod-SizeSelect-After.png|After Image | |||
</gallery> | |||
<li>Centrifuge tubes at 12000rpm for 1.5 minutes</li> | |||
<li>Discard 0.5 mL tube and resuspend in 500 uL TE buffer</li> | |||
<li>Incubate at 37C overnight with vigorous shaking; continued [[Daniel:Notebook/ComboLock/2016-9-29|tomorrow]]</li> | |||
</ol></ol> | |||
[[Category:ComboLock]] [[Category:20160928]] |
Latest revision as of 17:31, 29 September 2016
Positive Control Amplicon Production (PCAmp4)[edit]
Protocol[edit]
- 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
- Size Select
- For sample lanes, mix 32 uL sample, 28 uL 6X loading dye and 100 uL TBE (160 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
- 2016-09-28-PCAmp4Prod-SizeSelect.png
Before Image
- 2016-09-28-PCAmp4Prod-SizeSelect-After.png
After Image
- Centrifuge tubes at 12000rpm for 1.5 minutes
- Discard 0.5 mL tube and resuspend in 500 uL TE buffer
- Incubate at 37C overnight with vigorous shaking; 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[edit]
Sample | ng/uL ssDNA |
C4-C2 Amplicon | 557.1 |