Matt:LabNotes/2014-7-17: Difference between revisions
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>Mzcai (Created page with "==Agi26k0gap Probe Production== ===Production PCR=== *Make Master Mix for 46 well (6 strips) *0 gap first round amplicon -> V6 primers {| {{table}} | align="center" style="b...") |
>Mzcai mNo edit summary |
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(7 intermediate revisions by the same user not shown) | |||
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98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold | 98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold | ||
[[File: |650px]] | [[File:2014-07-17_Agi26k0gapProductionPCR.JPG|650px]] | ||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
*6 | *6 15-ml tubes (with 8 wells of PCR product each) for 0gap | ||
**800ul PCR product | **800ul PCR product | ||
**2000ul 100% EtOH | **2000ul 100% EtOH | ||
Line 36: | Line 36: | ||
**80ul 3M NaOAc pH 5.2-5.5 | **80ul 3M NaOAc pH 5.2-5.5 | ||
*Vortexed and put in -80C for | *Vortexed and put in -80C for overnight | ||
*Centrifuged at 3000rpm at 4C for | *Centrifuged at 3000rpm at 4C for 30min | ||
*Discarded supernatant and added 800ul of cold 80% EtOH | *Discarded supernatant and added 800ul of cold 80% EtOH | ||
*Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes | *Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes | ||
Line 48: | Line 48: | ||
*Eluted each column with 40ul and combined all probes into one 1.5ml | *Eluted each column with 40ul and combined all probes into one 1.5ml | ||
*Measured concentration with Nanodrop: | *Measured concentration with Nanodrop: | ||
*~220ul of Agi26k0gap probes: 352.2 ng/ul => 77.5 ug | |||
**Every time I tried to measure sample it gave me a warning message that there might be a bubble or unclean pedastal, but consistently measured ~355ng/ul (all 3 times) | |||
**In between the 3 sample measurements I cleaned and measured a blank water sample and got +/-2ng/ul with no warning message | |||
***Next day re-measured and got 355.5 ng/ul with no error | |||
===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== | ||
*Divide into | *Divide into eight pcr tubes of 27.5ul with total amplicon of ~10ug each | ||
**Four with old Lambda and Four with new Lambda | |||
***Fourth tube of each set had extra 2.5ul amplicon | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| Amplicon|| | | Amplicon||27.5 | ||
|- | |- | ||
| 10X Lambda Exo Buffer||10 | | 10X Lambda Exo Buffer||10 | ||
Line 63: | Line 68: | ||
| Lambda Exonuclease||10 | | Lambda Exonuclease||10 | ||
|- | |- | ||
| H2O|| | | H2O||52.5 | ||
|- | |- | ||
| Total||100 | | Total||100 | ||
Line 69: | Line 74: | ||
*Incubated at 37C for 1hr | *Incubated at 37C for 1hr | ||
*Purified | *Purified with of Zymo ssDNA/RNA columns | ||
*Eluted with | *Eluted with 40ul each | ||
*Combined the three tubes with 27.5ul amplicon to measure and compare | |||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
** | **New Lambda: 69.5ng/ul x 120ul = 8.34ug | ||
** | **Old Lambda: 65.3ng/ul x 120ul = 7.84ug | ||
**Zymo | ***Estimate total yield: ~67ng/ul x 320ul = 21.44ug (21.44/38.75 = 55.3%) | ||
*'''Result''': Negligible difference between old and new lambda | |||
**Not sure what was causing low yield during Lambda digestion [[Matt:LabNotes/2014-6-24]] | |||
**Checked Qiagen vs Zymo columns and that wasn't the reason either | |||
***Whatever it was, things seem to work well now | |||
* | *Continued on: [[Matt:LabNotes/2014-7-22]] |
Latest revision as of 18:23, 22 July 2014
Agi26k0gap Probe Production[edit]
Production PCR[edit]
- Make Master Mix for 46 well (6 strips)
- 0 gap first round amplicon -> V6 primers
Components | 1x rxn (ul) | 50x rxn (ul) |
First round amplicon (10nM) | 0.2 | 10 |
2x Kapa SYBR MM | 50 | 2500 |
100uM pAP1V6U | 0.4 | 20 |
100uM AP2V6 | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold
File:2014-07-17 Agi26k0gapProductionPCR.JPG
EtOH Precipitation[edit]
- 6 15-ml tubes (with 8 wells of PCR product each) for 0gap
- 800ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 80ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight
- Centrifuged at 3000rpm at 4C for 30min
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes
- Centrifuged 1.5ml tubes at 15,000rpm for 5 min on table-top centrifuge
- Discarded supernatant and air-dried for 5 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification[edit]
- Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
- Eluted each column with 40ul and combined all probes into one 1.5ml
- Measured concentration with Nanodrop:
- ~220ul of Agi26k0gap probes: 352.2 ng/ul => 77.5 ug
- Every time I tried to measure sample it gave me a warning message that there might be a bubble or unclean pedastal, but consistently measured ~355ng/ul (all 3 times)
- In between the 3 sample measurements I cleaned and measured a blank water sample and got +/-2ng/ul with no warning message
- Next day re-measured and got 355.5 ng/ul with no error
Lambda Exonuclease Digestion[edit]
- Divide into eight pcr tubes of 27.5ul with total amplicon of ~10ug each
- Four with old Lambda and Four with new Lambda
- Fourth tube of each set had extra 2.5ul amplicon
- Four with old Lambda and Four with new Lambda
Components | Volume |
Amplicon | 27.5 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 52.5 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with of Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Combined the three tubes with 27.5ul amplicon to measure and compare
- Recombined and measured ssDNA with Nanodrop:
- New Lambda: 69.5ng/ul x 120ul = 8.34ug
- Old Lambda: 65.3ng/ul x 120ul = 7.84ug
- Estimate total yield: ~67ng/ul x 320ul = 21.44ug (21.44/38.75 = 55.3%)
- Result: Negligible difference between old and new lambda
- Not sure what was causing low yield during Lambda digestion Matt:LabNotes/2014-6-24
- Checked Qiagen vs Zymo columns and that wasn't the reason either
- Whatever it was, things seem to work well now
- Continued on: Matt:LabNotes/2014-7-22