Matthew Cai:LabNotes/Probe Production and Capture: Difference between revisions
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>Mzcai (Created page with "==Experimental Plan== ===Oligo Resuspension and Expansion=== * After receiving the probes, resuspend with H2O to the concentration 20 nM (in case it is lyophilized) * perform...") |
>Mzcai |
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==Experimental Plan== | ==Experimental Plan== | ||
===Oligo Resuspension | ===Oligo Resuspension=== | ||
* After receiving the probes, resuspend with H2O to the concentration 20 nM (in case it is lyophilized) | * After receiving the probes, resuspend with H2O to the concentration 20 nM (in case it is lyophilized) | ||
* | * Perform expansion PCR to amplify the oligoes as the template | ||
===Oligo Expansion=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Final conc.''' | |||
|- | |||
| 20nM LC Sciences Oligoes||10.00||1nM | |||
|- | |||
| eMIP_CA1_F (100uM)||0.80||400nM | |||
|- | |||
| eMIP_CA1_R (100uM)||0.80||400nM | |||
|- | |||
| 2x Kapa SYBG MM||100.00||1x | |||
|- | |||
| H2O||88.40|| | |||
|- | |||
| Total||200.00|| | |||
|} | |||
'''Program'''<br> | |||
95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x '''5''' -> (95C 5sec -> 60C 30sec-> 72C 30sec) x '''10''' -> 72C 2min -> 15C hold | |||
* Primer info. | |||
** eMIP_CA1_F: TGCCT'''AGGACCGGATCAACT''' (TGCCT overhang), Tm of the short one = 53.06C, the long one = 63.28C | |||
** eMIP_CA1_R: GAGCT'''TCGGTTCACGCAATG''' (GAGCT overhang), Tm of the short one = 54.41C, the long one = 62.95C | |||
* Note: | |||
** The first five cycles, Tm of each primer was calculated without 5 nt overhang, then the Tm was increased based on the Tm of the full length primer | |||
** The number of cycles will be monitored | |||
* Purified with Qiaquick column (2 columns) and elute with EB buffer volume 50ul each --> total volume = 100ul | |||
* Measure DNA conc. with Nanodrop: 18ng/ul or 295.5nM | |||
* Dilute 1st round amplicon to 10nM volume 1000ul (mix 33.89ul of 295.5nM 1st round amplicon with 966.11ul H2O)--> for using as the template for the future amplification | |||
===Production PCR=== |
Revision as of 00:49, 27 February 2013
Experimental Plan
Oligo Resuspension
- After receiving the probes, resuspend with H2O to the concentration 20 nM (in case it is lyophilized)
- Perform expansion PCR to amplify the oligoes as the template
Oligo Expansion
Components | Volume (ul) | Final conc. |
20nM LC Sciences Oligoes | 10.00 | 1nM |
eMIP_CA1_F (100uM) | 0.80 | 400nM |
eMIP_CA1_R (100uM) | 0.80 | 400nM |
2x Kapa SYBG MM | 100.00 | 1x |
H2O | 88.40 | |
Total | 200.00 |
Program
95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x 5 -> (95C 5sec -> 60C 30sec-> 72C 30sec) x 10 -> 72C 2min -> 15C hold
- Primer info.
- eMIP_CA1_F: TGCCTAGGACCGGATCAACT (TGCCT overhang), Tm of the short one = 53.06C, the long one = 63.28C
- eMIP_CA1_R: GAGCTTCGGTTCACGCAATG (GAGCT overhang), Tm of the short one = 54.41C, the long one = 62.95C
- Note:
- The first five cycles, Tm of each primer was calculated without 5 nt overhang, then the Tm was increased based on the Tm of the full length primer
- The number of cycles will be monitored
- Purified with Qiaquick column (2 columns) and elute with EB buffer volume 50ul each --> total volume = 100ul
- Measure DNA conc. with Nanodrop: 18ng/ul or 295.5nM
- Dilute 1st round amplicon to 10nM volume 1000ul (mix 33.89ul of 295.5nM 1st round amplicon with 966.11ul H2O)--> for using as the template for the future amplification