Noi/NOTES/2011-10-6: Difference between revisions
Jump to navigation
Jump to search
>Noi |
>Noi |
||
Line 76: | Line 76: | ||
** measure conc. with Nanodrop --> 88.1ng/ul, yield ~17.6ng. The yield seems low when compare to the DMR330k preparation (ave ~30ug for 32 rxn, started with the same amount of template), but in this batch I stopped the reaction earlier (13cycles) and the size of product is smaller. | ** measure conc. with Nanodrop --> 88.1ng/ul, yield ~17.6ng. The yield seems low when compare to the DMR330k preparation (ave ~30ug for 32 rxn, started with the same amount of template), but in this batch I stopped the reaction earlier (13cycles) and the size of product is smaller. | ||
== Nicking | == Nicking endonuclease digestion == | ||
* Incubate 4ug of amplified amplicon with 10ul of Nt.AlwI (10U/ul) in total volume 200ul at 37C for 1hr (4x reactions) | * Incubate 4ug of amplified amplicon with 10ul of Nt.AlwI (10U/ul) in total volume 200ul at 37C for 1hr (4x reactions) | ||
* Heat inactivate enzyme at 80C for 20 min | * Heat inactivate enzyme at 80C for 20 min | ||
Line 84: | Line 84: | ||
* Before doing PAGE size selection to purify the 70nt band, I ran the gel to check if DNA nicking work well by loading the same amount of DNA before and after incubation with each enzyme at the same amount | * Before doing PAGE size selection to purify the 70nt band, I ran the gel to check if DNA nicking work well by loading the same amount of DNA before and after incubation with each enzyme at the same amount | ||
[[File:LC-probe-Nicking_2011_10_07.png| 280px]]<br> | [[File:LC-probe-Nicking_2011_10_07.png| 280px]]<br> | ||
* A = amplified amplicon before nicking | * A = amplified amplicon before nicking endonuclease digestion | ||
* B = After incubation with Nt.AlwI | * B = After incubation with Nt.AlwI | ||
* C = After incubation with Nb. BsrDI | * C = After incubation with Nb. BsrDI |
Revision as of 18:20, 8 October 2011
Plan of probe (from LC Bioscience) production
- Follow the protocol: [1] and standard protocol using to make DMR330k probe set
- After receiving the probes, resuspend with H2O to the concentration 20 nM (in case it is lyophilized)
- perform expansion PCR to amplify the oligoes as the template
- 2011_10_07 received oligo mix from LC Science
- Oligo info.: DNA oligo mix order 4617, concentration 14.94ng/ul-->490.65nM (ssDNA, size 100nt), volume 25ul
- Dilute to 20nM volume 100ul by mixing 4.08ul of oligo with 95.92ul H2O
Expansion PCR
Components | Volume (ul) | Final conc. |
20nM LC Sciences Oligoes | 10.00 | 1nM |
eMIP_CA1_F (100uM) | 0.80 | 400nM |
eMIP_CA1_F (100uM) | 0.80 | 400nM |
2x Kapa SYBG MM | 100.00 | 1x |
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
- Primer info.
- eMIP_CA1_F: TGCCTAGGACCGGATCAACT (TGCCT overhang), Tm of the short one = 53.06C, the long one = 63.28C
- eMIP_CA1_R: GAGCTTCGGTTCACGCAATG (GAGCT overhang), Tm of the short one = 54.41C, the long one = 62.95C
- Note:
- The first five cycles, Tm of each primer was calculated without 5 nt overhang, then the Tm was increased based on the Tm of the full length primer
- The number of cycles will be monitored
File:Expansion-PCR 2011 10 07.png
- Purified with Qiaquick column (2 columns) and elute with EB buffer volume 50ul each --> total volume = 100ul
- Measure DNA conc. with Nanodrop: 18ng/ul or 295.5nM
- Dilute 1st round amplicon to 10nM volume 1000ul (mix 33.89ul of 295.5nM 1st round amplicon with 966.11ul H2O)--> for using as the template for the future amplification
- Perform production PCR
Production PCR
Components | 1 rxn | 32x rxn mix |
1st round amplicon (10nM) | 0.20 | 6.40 |
eMIP_CA1_F (100uM) | 0.40 | 12.80 |
eMIP_CA1_R (100uM) | 0.40 | 12.80 |
2x Kapa SYBG MM | 50.00 | 1600.00 |
H2O | 49.00 | 1568.00 |
Total | 100.00 | 3200.00 |
Program
95C 30sec -> (95C 5sec -> 60C 30sec-> 72C 20sec) x 13 -> 72C 2min -> 15C hold
- The number of cycles will be monitored to prevent overamplification
File:ProductionPCR-LC 2011 10 07.png
- Dr. Zhang suggested to check the oligoes by running 2ul oligoes before amplification in 6% TBU gel and quantify concentration if it correspond to the concentration specified on the tube. Also run the 1st round amplicon (after column purification) and amplified amplicon (before purification)
- A1 = single strand LC oligoes before amplification 1.8ul
- B1 = 1st round amplicon after column purification 1.8ul (total volume 100ul)
- C1 = amplified amplicon before EtOH precipitation & column purification 1.8ul
- Note: A2, B2 & C2 = load sample 0.2ul to prevent intensity saturation when do quantification (haven't done yet)
- From the gel image, the concentration of oligoes from LC Sciences may be overestimated since it showed a very faint band at the 100 bp band (the expected amount of 1.8ul ~ 25ng), and I also observed the smear band along the lane. For the 1st round amplicon and amplified amplicon, they showed the specific band ~110bp as expected.
- Perform EtOH precipitation of the 32 rxn PCR in 4x 15mL tube
- 800ul amplified amplicon +80ul 3M NaOAc+2.6ul Glycoblue and 2mL 100EtOH
- incubate at -80C for 30min --> centrifuge at 3,000 rpm for 30 min, wash with 75% EtOH
- resuspend with 100ul each tube
- re-purify with Qiaquick column (4 columns) and elute with 50ul EB buffer each --> total volume 200ul purified amplicon
- measure conc. with Nanodrop --> 88.1ng/ul, yield ~17.6ng. The yield seems low when compare to the DMR330k preparation (ave ~30ug for 32 rxn, started with the same amount of template), but in this batch I stopped the reaction earlier (13cycles) and the size of product is smaller.
Nicking endonuclease digestion
- Incubate 4ug of amplified amplicon with 10ul of Nt.AlwI (10U/ul) in total volume 200ul at 37C for 1hr (4x reactions)
- Heat inactivate enzyme at 80C for 20 min
- Add 10ul Nb.BrsDI (10U/ul) at 65C for 1 hr
- Purify with 6 Qiaquick colums, elute with 50ul EB buffer each --> total volume 300ul (Note: use more Qiaquick columns since the volume of of reaction was large, total 800ul and doing multiple pass of DNA through the column didn't help to recover DNA very well)
- Measure the conc. of nicked DNA with Nanodrop -->50.4ng/ul, yields = 15.1ug (why recovery rate so high > 85%?)
- Before doing PAGE size selection to purify the 70nt band, I ran the gel to check if DNA nicking work well by loading the same amount of DNA before and after incubation with each enzyme at the same amount
File:LC-probe-Nicking 2011 10 07.png
- A = amplified amplicon before nicking endonuclease digestion
- B = After incubation with Nt.AlwI
- C = After incubation with Nb. BsrDI
- Note: The digestion was not very complete since I still see the partial digested band of ~ 90nt. I think that the condition using to nick amplified amplicon may not be very optimum since the amount of the input DNA is high and I try to minimize the volume of reaction. However in this experiment I have no time to optimize and I think that the expected band (~70nt or slightly lower) was a lot brighter than the incomplete digested band, so I continued to do PAGE size-selection to purify the 70nt band in 6% TBU gel.
- PAGE quantification will be performed to check the efficiency of each enzyme by comparing the ratio of the intensity of each band.
File:ZhangLab 2 2011-10-08 00hr 11min PAGE-SS-1.jpgFile:ZhangLab 2 2011-10-08 00hr 15min PAGE-SS-2.jpg
- I used the old TBU gel prepared 2 weeks and almost 1 month before, the resolution of the DNA was not very good. The gel prepare 2 week gave the a better resolution the 1 month prepared gel. Would recommend to use the fresh prepared gel or not > 2 weeks if there is no time restriction.
- I incubated the gel eluted DNA overnight with EtOH, NaOAc, and Glycoblue at -80 --> will precipitate and quantify concentration on Saturday 08, 2011
Plan for Saturday
Capture set up
- Perform bisulfite conversion of PGP1F, PGP1iPS, and H1 to be enough for 4 capture reactions each (to optimize annealing temperature). Re-measure gDNA conc. before bisulfite conversion.
- Bis-cvt DNA ~ 300ng/rxn if it's enough (may use 250ng if the total yield is not enough)
- Probe/ target ratio: 1,000/1 (since the probe size is very small (4,000), to increase capture efficiency, Dr. Zhang suggested to increase the probe to target ratio).
Probe calculation
Probe/target | 1000:1 | ' |
Probe size | 4,000 | |
Template | 300.00 | ng |
Human gDNA MW | 1.820E+12 | g/mole |
Human gDNA (300ng) | 1.648E-19 | mole |
Probe required | 1.648E-16 | mole |
Probe MW | 8.649E+07 | g/mole (4,000x70bpx308.9D/bp+79D ) |
Amount probe required | 1.426E-08 | g |
Amount probe required | 14.26 | ng |
- Expected probe amount required for ~12-15 rxn = 214ng
Capture plan
- Capture volume --> in 15-20ul
- Vary annealing temperature --> try all below 55C we are currently using for DMR330k BSPP capture (since the probe size is shorter) --> 47.5C, 50.0C, 52.5C, 55.0C ??? please suggest
Program
95c 30sec -> cool down to X C at 0.02C/sec -> X C 20h
-> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP)
-> X C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
- Note: I try not to change the capture condition this time since want to make sure that everything will work as in the past except varying the annealing temp.