Matt:LabNotes/2016-7-26: Difference between revisions
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>Mzcai |
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===Qia Column Purification=== | ===Qia Column Purification=== | ||
*8 columns elute 50ul each | *8 columns elute 50ul each | ||
*Nanodrop: | *Nanodrop: 374.4 ng/uL x 400uL = 149.76ug | ||
===Lambda Exo=== | ===Lambda Exo=== |
Revision as of 19:04, 27 July 2016
TB12k_Apr2016 V4 Probe Preparation
Production PCR
- Make 10ml V4 master mix for 96 wells
Components | Volume (1X) | Volume (100X) |
First round amplicon TB12k_Apr2016_V4 (10nM) | 0.2 | 20 |
2X KAPA SYBG MM | 50 | 5000 |
AP1V4U (100uM) | 0.4 | 40 |
AP2V4 (100uM) | 0.4 | 40 |
H2O | 49 | 4900 |
Total | 100 | 10,000 |
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 4C hold
- Did in regular thermocyclers
EtOH Precipitation
- 8 15-ml tubes (with 12 wells (1.5 strips) of PCR product each) for
- 1,200ul PCR product
- 3000ul 100% EtOH
- 4ul GlycoBlue
- 120ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 30 min
- Centrifuged at 3000rpm at 4C for 30 min
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes
- Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
- Discarded supernatant and air-dried for 5 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification
- 8 columns elute 50ul each
- Nanodrop: 374.4 ng/uL x 400uL = 149.76ug
Lambda Exo
- Divide into 8 pcr tubes of 100ul (ideally should be <10ug each)
Components | Volume |
Amplicon | 50 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 30 |
Total | 100 |
- Incubated at 37C for 2hr (normally 1hr)