Noi/NOTES/2013-3-6: Difference between revisions

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* Aliquot 22.5ul, add 2.5ul of DNA template
* Aliquot 22.5ul, add 2.5ul of DNA template
* Program
'''Program'''<br>
95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x 5 -> (95C 5sec -> 60C 30sec-> 72C 30sec) x 10 -> 72C 2min -> 15C hold<br>
95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x 5 -> (95C 5sec -> 60C 30sec-> 72C 30sec) x 10 -> 72C 2min -> 15C hold<br>
* Verify the PCR product and also the seed oligos from original tube
* Verify the PCR product and also the seed oligos from original tube
* If everything works well, repeat expansion PCR in total volume 200ul
* If everything works well, repeat expansion PCR in total volume 200ul
== Expansion PCR ==
== Expansion PCR ==
{| {{table}}
{| {{table}}
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| Total||200.00
| 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  
'''Program'''<br>
95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x 5 -> (95C 5sec -> 60C 30sec-> 72C 30sec) x 10 -> 72C 2min -> 15C hold<br>
  - Purified with Qiaquick column (2 columns) and elute with EB buffer volume 50ul each --> total volume = 100ul
  - Purified with Qiaquick column (2 columns) and elute with EB buffer volume 50ul each --> total volume = 100ul
  - Measure DNA conc. with Nanodrop: ng/ul or nM
  - Measure DNA conc. with Nanodrop: ng/ul or nM
  - Dilute 1st round amplicon to 10nM volume XX ul (mix ul of nM 1st round amplicon with ul H2O)--> for using as the template for the future amplification
  - Dilute 1st round amplicon to 10nM volume XX ul (mix ul of nM 1st round amplicon with XX ul H2O)
--> for using as the template for the future amplification
  - Perform production PCR
  - Perform production PCR
== Production PCR ==
== Production PCR ==
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* Aliquot 100ul to 3 8-striptubes
* Aliquot 100ul to 3 8-striptubes
'''Program'''<br>
95C 30sec -> (95C 5sec -> 60C 30sec-> 72C 30sec) x 13 -> 72C 2min -> 15C hold<br>
* Note:
** I increased extension time to 30sec)
** Number of cycle will be monitor (might set at 15 cycles)

Revision as of 22:58, 6 March 2013

Plan for probe preparation for Matt's project

  • Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-2-27#Plan_for_probe_preparation_for_Matt.27s_project
  • 2013/03/06: received seed oligos from CustomArray
  • Seed oligo information
    • 200 nt
    • Total amount 4.95ug (61.93ng/ul in total volume 80ul in TE buffer
  • Calculate MW of 200 nt probe = 200nt * 308.9D/bp + 79D = 61859 g/mol
  • 61.93ng/ul = 1.00114 ~ 1 uM or 1000 nM --> calculated from (61.93ng/ul/61,859g/mol) need to convert the unit to mol/L at the end
  • Aliquot 2ul of 61.93ng/ul oligoes for PAGE verification & save original tube with Matt
  • Since the concentration is quit high, I will dilute to 100nM first (in 100ul) then dilute to 20uM (in 200ul)
- To dilute from 1uM to 100nM volume 100ul: mix 10ul of 61.93 ng/ul oligos (or 1uM) with 90ul H2O
- To dilute from 100nM to 20nM volume 200: mix 40ul of 100nM oligos with 160ul H2O
  • Test if the PCR works in small volume (50ul) by using LC Sciences oligoes as +ve control
    • 1. NTC
    • 2. Positive control (LC Sciences oligos)
    • 3. 12k CustomArray oligos
Components Volume (ul) Final conc. Volume for 3.5 rxn mix
20nM seed oligoes 2.50 1nM 0.00
eMIP_CA1_F (100uM) 0.20 400nM 0.70
eMIP_CA1_R (100uM) 0.20 400nM 0.70
2x KAPA SYBG fast MM 25.00 1x 87.50
H2O 22.10 77.35
Total 50.00 175.00
  • Aliquot 22.5ul, add 2.5ul of DNA template

Program
95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x 5 -> (95C 5sec -> 60C 30sec-> 72C 30sec) x 10 -> 72C 2min -> 15C hold

  • Verify the PCR product and also the seed oligos from original tube
  • If everything works well, repeat expansion PCR in total volume 200ul

Expansion PCR

Components Volume (ul)
20nM seed oligoes 10.00
eMIP_CA1_F (100uM) 0.80
eMIP_CA1_R (100uM) 0.80
2x KAPA SYBG fast MM 100.00
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

- Purified with Qiaquick column (2 columns) and elute with EB buffer volume 50ul each --> total volume = 100ul
- Measure DNA conc. with Nanodrop: ng/ul or nM
- Dilute 1st round amplicon to 10nM volume XX ul (mix ul of nM 1st round amplicon with XX ul H2O)
--> for using as the template for the future amplification
- Perform production PCR

Production PCR

Components 1 rxn 25x rxn mix
1st round amplicon (10nM) 0.20 5.00
eMIP_CA1_F (100uM) 0.40 10.00
eMIP_CA1_R (100uM) 0.40 10.00
2x KAPA SYBG fast MM 50.00 1,250.00
H2O 49.00 1,225.00
Total 100.00 2,500.00
  • Aliquot 100ul to 3 8-striptubes

Program
95C 30sec -> (95C 5sec -> 60C 30sec-> 72C 30sec) x 13 -> 72C 2min -> 15C hold

  • Note:
    • I increased extension time to 30sec)
    • Number of cycle will be monitor (might set at 15 cycles)