Noi/NOTES/2011-10-6: Difference between revisions
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== Plan of probe (from LC | == Plan of probe (from LC Science) production == | ||
* Follow the protocol: [http://arep.med.harvard.edu/pdf/Porreca07.pdf] and standard protocol using to make DMR330k probe set<br> | * Follow the protocol: [http://arep.med.harvard.edu/pdf/Porreca07.pdf] and standard protocol using to make DMR330k probe set<br> | ||
* After receiving the probes, resuspend with H2O to the concentration 20 nM (in case it is lyophilized) | * After receiving the probes, resuspend with H2O to the concentration 20 nM (in case it is lyophilized) | ||
Line 18: | Line 18: | ||
| eMIP_CA1_F (100uM)||0.80||400nM | | eMIP_CA1_F (100uM)||0.80||400nM | ||
|- | |- | ||
| | | eMIP_CA1_R (100uM)||0.80||400nM | ||
|- | |- | ||
| 2x Kapa SYBG MM||100.00||1x | | 2x Kapa SYBG MM||100.00||1x | ||
Line 74: | Line 74: | ||
** resuspend with 100ul each tube | ** resuspend with 100ul each tube | ||
** re-purify with Qiaquick column (4 columns) and elute with 50ul EB buffer each --> total volume 200ul purified amplicon | ** 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. | ** measure conc. with Nanodrop --> 88.1ng/ul, yield ~17.6ug. 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 ( | * 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 | ||
* Add 10ul Nb. | * Add 10ul Nb.BsrDI (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%?) | * 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 | * 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 | ||
* Note: The digestion was not very complete since I still see the partial digested band of ~ | * 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. | * 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.jpg| 350px]][[File:ZhangLab_2 2011-10-08 00hr 15min_PAGE-SS-2.jpg| 380px]]<br> | [[File:ZhangLab_2 2011-10-08 00hr 11min_PAGE-SS-1.jpg| 350px]][[File:ZhangLab_2 2011-10-08 00hr 15min_PAGE-SS-2.jpg| 380px]]<br> | ||
* I used the old TBU gel prepared 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. | * 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 | |||
* After EtOH precipatition and washing, resuspend the probe with 15ul each tube --> total volume 150ul | |||
* PAGE quantification in 6% TBU gel (mix 2ul of probe in total 80 u and load different volume to estimate DNA concemtration) | |||
[[File:LC-probe-PQ-1.png| 200px]]<br> | |||
* Final conc. = 4.10ng/ul --> total tields = 150ulx4.1ng/ul = 615ng (lower than expected) | |||
== 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 == | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Probe/target''' | |||
| align="center" style="background:#f0f0f0;"|'''1000:1''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| 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) --> 48C, 50, 52C, 55C | |||
'''Program'''<br> | |||
95c 30sec -> cool down to 48-54 C at 0.02C/sec -> X C 17.5h<br> | |||
-> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP)<br> | |||
-> 48-54 C 17h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.<br> | |||
* Note: I try not to change the capture condition this time since want to make sure that everything will work well as in the past except varying the annealing temp. | |||
== Amplification == | |||
* To test whether the capture work and what is the best annealing temperature for this probe set, I ran the PCR in small volume 25ul and using 2ul of circularized DNA template. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''1 rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''14rxn mix''' | |||
|- | |||
| Captured DNA template||2.00||0.00 | |||
|- | |||
| 10uM CP-2-FA||0.50||7.00 | |||
|- | |||
| 10uM CP-2-RA||0.50||7.00 | |||
|- | |||
| 2xKAPA MM||12.50||175.00 | |||
|- | |||
| H2O||9.50||133.00 | |||
|- | |||
| Total||25.00||350.00 | |||
|} | |||
* Aliquot 23ul, add 2ul of DNA template | |||
'''Program'''<br> | |||
98C 30sec--> (98C 10sec --> 52C 30sec --> 72C 30sec)x5 cycles --> (98C 10sec --> 60C 30sec --> 72C 30sec)x15 cycles--> 72C 3 min --> 15C hold<br> | |||
[[File:2011_10_10-test capture20cycles.png| 700px]]<br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Samples''' | |||
| align="center" style="background:#f0f0f0;"|'''Ct 48C''' | |||
| align="center" style="background:#f0f0f0;"|'''Ct 50C''' | |||
|- | |||
| PGP1F||16.77||16.92 | |||
|- | |||
| PGP1iPS||16.72||17.12 | |||
|- | |||
| H1||16.51||16.81 | |||
|}<br> | |||
* To verify the PCR product, 2 ul of each samples were loaded in 6% TBE gel | |||
[[File:ZhangLab_2 2011-10-10 17hr 30min_Amp-CP-2.jpg| 500px]] | |||
* The expected band of amplicon ~ 260bp. Since there were many bands appeared in the gel, Dr. Zhang suggested to do PAGE size-selection to cut the 260bp band before doing PCR with the barcoded primers. | |||
* I repeated the amplification again in total volume 100ul by using the circularized DNA from 50, 52 and 54C annealing temperature and purified with Ampure beads (eluted volume 50ul). However, I used only the samples from 52 and 54C in PAGE size-selection and the next step. | |||
[[File:ZhangLab_2 2011-10-11 00hr 03min-PAGE-SS-PGP1F.jpg| 320px]][[File:ZhangLab_2 2011-10-10 23hr 57min-PAGE-SS-PGP1iPS.jpg| 320px]] [[File:ZhangLab_2 2011-10-10 23hr 55min-PAGE-SS-H1.jpg| 320px]]<br> | |||
* After EtOH precipitation I resuspended each samplle with 12ul H2O. | |||
== Amplification with adapter primers == | |||
* Reaction #1: CP-2-FA.IndSol (Indx =7, 45, and 76) & CP-2-RA.Sol | |||
* Reaction #2: Switch.CP-2-FA.Sol & Switch.CP-2-RA.IndSol (Indx = 7, 45, and 76) | |||
* I did the reaction #1 first to see if the amplification work. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''1 rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''7.5 rxn mix''' | |||
|- | |||
| template||4.00||0.00 | |||
|- | |||
| 10uM CA-2-FA.Indx Sol||2.00||0.00 | |||
|- | |||
| 10uM CA-2-RA.Sol||2.00||15.00 | |||
|- | |||
| 2x KAPA MM||50.00||375.00 | |||
|- | |||
| H2O||42.00||315.00 | |||
|- | |||
| Total||100.00||750.00 | |||
|} | |||
* Aliquot 94ul, add 4 ul template and 2ul Indx primer | |||
'''Program'''<br> | |||
98C 30sec --> (98 C 10 sec --> 54C 30sec --> 72C 30sec) x 4 --> 72 C 3 min<br> | |||
* For both FA.IndSol or RA.IndSol, each sample used the same index number. | |||
[[File:reaction1.png| 500px]]<br> | |||
* Reaction #2 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''1 rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''7.5 rxn mix''' | |||
|- | |||
| template||4.00||0.00 | |||
|- | |||
| 10uM Switch.CA-2-FA.Sol||2.00||15.00 | |||
|- | |||
| 10uM Switch.CA-2-RA.IndSol||2.00||0.00 | |||
|- | |||
| 2x KAPA MM||50.00||375.00 | |||
|- | |||
| H2O||42.00||315.00 | |||
|- | |||
| Total||100.00||750.00 | |||
|} | |||
Aliquot 94ul, add 4 ul template and 2ul Indx primer | |||
* Used the same PCR cycler as reaction #1 | |||
[[File:reaction2.png| 500px]]<br> | |||
* Purified PCR product with Ampure bead and eluted with 40ul EB buffer | |||
* Perform PAGE quantification by loading 1ul of purified amplicon in 6% TBE gel. I also loaded the size-selected DNA used as the template to see if I cut the right band. | |||
[[File:ZhangLab_2 2011-10-11 05hr 37min-PQ-1.jpg| 400px]][[File:ZhangLab_2 2011-10-11 05hr 40min-PQ-2.jpg| 400px]]<br> | |||
* 1 = PGP1 F 52C, 2 = PGP1F 54C, 3 = PGP1iPS 52C, 4 = PGP1iPS 54C, 5 = H1 52C, 6 = H1 54C<br> | |||
* A = PGP1F, B = PGP1iPS, C = H1<bR> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample /Reaction#1''' | |||
| align="center" style="background:#f0f0f0;"|'''conc. (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''yields in 40ul (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume for 25 ng''' | |||
|- | |||
| PGP1F 52C ||1.56||62.55||15.99 | |||
|- | |||
| PGP1F 54C||0.90||35.88||27.87 | |||
|- | |||
| PGP1iPS 52C ||1.61||64.40||15.53 | |||
|- | |||
| PGP1iPS 54C||1.08||43.33||23.08 | |||
|- | |||
| H1 52C||3.64||145.51||6.87 | |||
|- | |||
| H1 54C||2.74||109.69||9.12 | |||
|- | |||
| | |||
|- | |||
| Sample /Reaction#2||conc. (ng/ul)||yields in 40ul (ng)||Volume for 25 ng | |||
|- | |||
| PGP1F 52C swi||0.82||32.62||30.65 | |||
|- | |||
| PGP1F 54C swi||0.61||24.36||41.06 | |||
|- | |||
| PGP1iPS 52C swi||0.64||25.76||38.81 | |||
|- | |||
| PGP1iPS 54C swi||0.97||38.81||25.76 | |||
|- | |||
| H1 52C swi||3.09||123.57||8.09 | |||
|- | |||
| H1 54C swi||2.15||86.02||11.63 | |||
|} | |||
* I pooled the samples with the same annealing temperature during capture together, so I would have the libraries from 52 and 54C | |||
* Did PAGE size-selection even I could see the clear band ~350bp since I still see a little smear below the expected band. | |||
[[File:ZhangLab_2 2011-10-11 07hr 27min-PAGE-SeqLib.jpg| 400px]]<br> | |||
* Resuspended DNA with H2O --> total 20ul, and quantified conc. by Qubit dsDNA HS | |||
* SampleID: NP-BSPP-LC-52C_Oct6, 2011: 2.2ng/ul --> yields = 20ul x 2.2ng/ul = 44ng, --> 10.3nM (calculated based on size 350bp) | |||
* SampleID: NP-BSPP-LC-54C_Oct6, 2011: 1.73ng/ul --> yields = 20ul x 1.73ng/ul = 34.6ng --> 8.1nM (calculated based on size 350bp) | |||
[[File:ZhangLab_2 2011-10-11 11hr 06min-SeqLib-PC.jpg| 300px]] |
Latest revision as of 00:43, 28 February 2013
Plan of probe (from LC Science) production[edit]
- 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[edit]
Components | Volume (ul) | Final conc. |
20nM LC Sciences Oligoes | 10.00 | 1nM |
eMIP_CA1_F (100uM) | 0.80 | 400nM |
eMIP_CA1_R (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[edit]
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 ~17.6ug. 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[edit]
- 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.BsrDI (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
- After EtOH precipatition and washing, resuspend the probe with 15ul each tube --> total volume 150ul
- PAGE quantification in 6% TBU gel (mix 2ul of probe in total 80 u and load different volume to estimate DNA concemtration)
- Final conc. = 4.10ng/ul --> total tields = 150ulx4.1ng/ul = 615ng (lower than expected)
Plan for Saturday[edit]
Capture set up[edit]
- 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[edit]
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[edit]
- 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) --> 48C, 50, 52C, 55C
Program
95c 30sec -> cool down to 48-54 C at 0.02C/sec -> X C 17.5h
-> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP)
-> 48-54 C 17h-> 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 well as in the past except varying the annealing temp.
Amplification[edit]
- To test whether the capture work and what is the best annealing temperature for this probe set, I ran the PCR in small volume 25ul and using 2ul of circularized DNA template.
Components | 1 rxn | 14rxn mix |
Captured DNA template | 2.00 | 0.00 |
10uM CP-2-FA | 0.50 | 7.00 |
10uM CP-2-RA | 0.50 | 7.00 |
2xKAPA MM | 12.50 | 175.00 |
H2O | 9.50 | 133.00 |
Total | 25.00 | 350.00 |
- Aliquot 23ul, add 2ul of DNA template
Program
98C 30sec--> (98C 10sec --> 52C 30sec --> 72C 30sec)x5 cycles --> (98C 10sec --> 60C 30sec --> 72C 30sec)x15 cycles--> 72C 3 min --> 15C hold
File:2011 10 10-test capture20cycles.png
Samples | Ct 48C | Ct 50C |
PGP1F | 16.77 | 16.92 |
PGP1iPS | 16.72 | 17.12 |
H1 | 16.51 | 16.81 |
- To verify the PCR product, 2 ul of each samples were loaded in 6% TBE gel
File:ZhangLab 2 2011-10-10 17hr 30min Amp-CP-2.jpg
- The expected band of amplicon ~ 260bp. Since there were many bands appeared in the gel, Dr. Zhang suggested to do PAGE size-selection to cut the 260bp band before doing PCR with the barcoded primers.
- I repeated the amplification again in total volume 100ul by using the circularized DNA from 50, 52 and 54C annealing temperature and purified with Ampure beads (eluted volume 50ul). However, I used only the samples from 52 and 54C in PAGE size-selection and the next step.
File:ZhangLab 2 2011-10-11 00hr 03min-PAGE-SS-PGP1F.jpgFile:ZhangLab 2 2011-10-10 23hr 57min-PAGE-SS-PGP1iPS.jpg File:ZhangLab 2 2011-10-10 23hr 55min-PAGE-SS-H1.jpg
- After EtOH precipitation I resuspended each samplle with 12ul H2O.
Amplification with adapter primers[edit]
- Reaction #1: CP-2-FA.IndSol (Indx =7, 45, and 76) & CP-2-RA.Sol
- Reaction #2: Switch.CP-2-FA.Sol & Switch.CP-2-RA.IndSol (Indx = 7, 45, and 76)
- I did the reaction #1 first to see if the amplification work.
Components | 1 rxn | 7.5 rxn mix |
template | 4.00 | 0.00 |
10uM CA-2-FA.Indx Sol | 2.00 | 0.00 |
10uM CA-2-RA.Sol | 2.00 | 15.00 |
2x KAPA MM | 50.00 | 375.00 |
H2O | 42.00 | 315.00 |
Total | 100.00 | 750.00 |
- Aliquot 94ul, add 4 ul template and 2ul Indx primer
Program
98C 30sec --> (98 C 10 sec --> 54C 30sec --> 72C 30sec) x 4 --> 72 C 3 min
- For both FA.IndSol or RA.IndSol, each sample used the same index number.
- Reaction #2
Components | 1 rxn | 7.5 rxn mix |
template | 4.00 | 0.00 |
10uM Switch.CA-2-FA.Sol | 2.00 | 15.00 |
10uM Switch.CA-2-RA.IndSol | 2.00 | 0.00 |
2x KAPA MM | 50.00 | 375.00 |
H2O | 42.00 | 315.00 |
Total | 100.00 | 750.00 |
Aliquot 94ul, add 4 ul template and 2ul Indx primer
- Used the same PCR cycler as reaction #1
- Purified PCR product with Ampure bead and eluted with 40ul EB buffer
- Perform PAGE quantification by loading 1ul of purified amplicon in 6% TBE gel. I also loaded the size-selected DNA used as the template to see if I cut the right band.
File:ZhangLab 2 2011-10-11 05hr 37min-PQ-1.jpgFile:ZhangLab 2 2011-10-11 05hr 40min-PQ-2.jpg
- 1 = PGP1 F 52C, 2 = PGP1F 54C, 3 = PGP1iPS 52C, 4 = PGP1iPS 54C, 5 = H1 52C, 6 = H1 54C
- A = PGP1F, B = PGP1iPS, C = H1
Sample /Reaction#1 | conc. (ng/ul) | yields in 40ul (ng) | Volume for 25 ng |
PGP1F 52C | 1.56 | 62.55 | 15.99 |
PGP1F 54C | 0.90 | 35.88 | 27.87 |
PGP1iPS 52C | 1.61 | 64.40 | 15.53 |
PGP1iPS 54C | 1.08 | 43.33 | 23.08 |
H1 52C | 3.64 | 145.51 | 6.87 |
H1 54C | 2.74 | 109.69 | 9.12 |
Sample /Reaction#2 | conc. (ng/ul) | yields in 40ul (ng) | Volume for 25 ng |
PGP1F 52C swi | 0.82 | 32.62 | 30.65 |
PGP1F 54C swi | 0.61 | 24.36 | 41.06 |
PGP1iPS 52C swi | 0.64 | 25.76 | 38.81 |
PGP1iPS 54C swi | 0.97 | 38.81 | 25.76 |
H1 52C swi | 3.09 | 123.57 | 8.09 |
H1 54C swi | 2.15 | 86.02 | 11.63 |
- I pooled the samples with the same annealing temperature during capture together, so I would have the libraries from 52 and 54C
- Did PAGE size-selection even I could see the clear band ~350bp since I still see a little smear below the expected band.
File:ZhangLab 2 2011-10-11 07hr 27min-PAGE-SeqLib.jpg
- Resuspended DNA with H2O --> total 20ul, and quantified conc. by Qubit dsDNA HS
- SampleID: NP-BSPP-LC-52C_Oct6, 2011: 2.2ng/ul --> yields = 20ul x 2.2ng/ul = 44ng, --> 10.3nM (calculated based on size 350bp)
- SampleID: NP-BSPP-LC-54C_Oct6, 2011: 1.73ng/ul --> yields = 20ul x 1.73ng/ul = 34.6ng --> 8.1nM (calculated based on size 350bp)