Revision as of 22:49, 6 March 2013 by >Noi
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 (10ul 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
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 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