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

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  [[File:2013_03_07_TestqPCR_12koligos_variedconc.png| 650px]]
  [[File:2013_03_07_TestqPCR_12koligos_variedconc.png| 650px]]
   
   
  '''Ct values'''
  '''Ct values'''  
  - +ve 1nM LC oligos =  
  - +ve 1nM LC oligos = 14.26
  - 5nM 12K oligos =  
  - 5nM 12K oligos = 17.08
  - 10nM 12K oligos =  
  - 10nM 12K oligos = 16.36
  - 20nM 12K oligos =  
  - 20nM 12K oligos = 15.46
* PAGE verification  
* PAGE verification
<br>
<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

Revision as of 00:21, 8 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: 61.93(ng/ul)/61,859(g/mol) need to convert the unit to mol/L at the end
  • Aliquot 2ul of 61.93ng/ul oligos 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 1uM oligos (61.93 ng/ul) with 90ul H2O
- To dilute from 100nM to 20nM volume 200ul: mix 40ul of 100nM oligos with 160ul H2O

  • 3/07/2013
    • After I diluted oligo to 100nM, I measured the oligo concentration by Nanodrop: 10.5 ng/ul (after dilute 10X, expected conc. ~ 6.1ng/ul)

Expansion PCR Test

  • 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 47.5ul, 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

Result

  • After I did 15 cycles of amplification, only reaction with LC sciences oligo works, but very small signal from 12k CustomArray oligos was observed (<500 fluorescent intensity, using Eppendorrf). This is possible that there are only 10-15% of the full-length in the oligo pools as Marcelo mentioned in email.
File:2013 03 07 TestqPCR 12koligos 1nM.png

Repeat Expansion PCR Test

  • I repeat amplification by increasing amount of oligos to 5X, 10X, and 20X, and used the 100nM oligos as the template. I am not sure if the high concentration of oligos including the truncated oligos will interfere the PCR or not
  • I also increase extension time to 45 sec even 30 sec is enough to synthesize 200nt DNA fragment, and increase the second step PCR to 15 cycles.
    • 1. NTC
    • 2. Positive control (LC Sciences oligos)
    • 3. 12k CustomArray oligos 5X (added 1.25ul of 100nM in 25ul reaction)
    • 4. 12k CustomArray oligos 5X (added 2.5ul of 100nM in 25ul reaction)
    • 5. 12k CustomArray oligos 5X (added 5ul of 100nM in 25ul reaction)
Components 1 rxn 5.5 rxn mix
20nM seed oligoes 1.25 0.00
eMIP_CA1_F (100uM) 0.10 0.55
eMIP_CA1_R (100uM) 0.10 0.55
2x Kapa SYBG MM 12.50 68.75
H2O 11.05 60.78
Total 25.00 137.50
  • Aliquot 23.75

Program
95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 45sec) x 5 -> (95C 5sec -> 60C 30sec-> 72C 45sec) x 15 -> 72C 2min -> 15C hold

Results

File:2013 03 07 TestqPCR 12koligos variedconc.png

Ct values 
- +ve 1nM LC oligos = 14.26
- 5nM 12K oligos = 17.08
- 10nM 12K oligos = 16.36
- 20nM 12K oligos = 15.46
  • PAGE verification


  • 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 monitored (might set at 15 cycles and stop once it reaches the peak)