Noi/NOTES/2013-3-6: Difference between revisions
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== Result == | == 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 Eppendorf). This is possible that there are only 10-15% of the full-length in the oligo pools as Marcelo mentioned in email. | * 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 Eppendorf). 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| 400px]] | |||
== Repeat amplification == | == Repeat amplification == | ||
* 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 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 | ||
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'''Program'''<br> | '''Program'''<br> | ||
95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 45sec) x 5 -> (95C 5sec -> 60C 30sec-> 72C 45sec) x 15 -> 72C 2min -> 15C hold<br> | 95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 45sec) x 5 -> (95C 5sec -> 60C 30sec-> 72C 45sec) x 15 -> 72C 2min -> 15C hold<br> | ||
== Results == | |||
[[File:2013_03_07_TestqPCR_12koligos_variedconc.png| 500px]] | |||
'''Ct values''' | |||
- +ve 1nM LC oligos = | |||
- 5nM 12K oligos | |||
- 10nM 12K oligos | |||
- 20nM 12K oligos | |||
<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 23:44, 7 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)
- 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 Eppendorf). 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 amplification
- 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 = - 5nM 12K oligos - 10nM 12K oligos - 20nM 12K oligos
- 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)