Noi/NOTES/2013-3-6: Difference between revisions
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* Calculate MW of 200 nt probe = 200nt * 308.9D/bp + 79D = 61859 g/mol | * 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 | * 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 | * 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) | * 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 1uM to 100nM volume 100ul: mix 10ul of 1uM oligos (61.93 ng/ul) with 90ul H2O | ||
- To dilute from 100nM to 20nM volume | - To dilute from 100nM to 20nM volume 200ul: 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 | * Test if the PCR works in small volume (50ul) by using LC Sciences oligoes as +ve control | ||
** 1. NTC | ** 1. NTC |
Revision as of 16:36, 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
- 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 monitored (might set at 15 cycles and stop once it reaches the peak)