Noi/NOTES/2013-3-6
Jump to navigation
Jump to search
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. +ve control 1nM LC Sciences oligos
- 3. 1nM 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. +ve control 1nM LC Sciences oligos
- 3. 5nM 12k CustomArray oligos (5X, added 1.25ul of 100nM in 25ul reaction)
- 4. 10nM 12k CustomArray oligos (10X, added 2.5ul of 100nM in 25ul reaction)
- 5. 20nM 12k CustomArray oligos (20X, 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
File:ZhangLab 2 2013-03-07 17hr 02min TestExpansionPCR variedconc.jpg
- Note: I loaded 2.5ul of PCR products in TBU gel, so the size might be slightly different compare to in TBE gel. However, from the gel image, the size of amplicons is very close to 200bp fragment.
- 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)