Noi/NOTES/2011-10-6: Difference between revisions

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== Plan of probe (from LC Bioscience) production ==
== 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_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 ~17.6ng. 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 ~17.6ug. 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 exonuclease digestion ==
== Nicking endonuclease digestion ==
* Incubate 4ug of amplified amplicon with 10ul of Nt.AlwI (10U/ul) in total volume 200ul at 37C for 1hr (4 tube reaction)
* 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.BrsDI (10U/ul) at 65C for 1 hr
* 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)
* 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 exonuclease digestion
* 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   
Line 92: Line 92:
* 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 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
* 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 ==
== Plan for Saturday ==
Line 120: Line 124:
|-
|-
| Amount probe required||14.26||ng
| Amount probe required||14.26||ng
|}<br>
|}
* Expected probe amount required for ~12-15 rxn = 214ng
* Expected probe amount required for ~12-15 rxn = 214ng
== Capture plan ==
== Capture plan ==
* Capture volume --> in 15-20ul  
* 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
* 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>
'''Program'''<br>
95c 30sec -> cool down to X C at 0.02C/sec -> X C 20h<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)  
-> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP)<br>
  -> X C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
-> 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 as in the past except varying the annealing temp.
* 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)

File:LC-PQ 2011 10 07.png

  • 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)

File:LC-probe-PQ-1.png

  • 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.

File:Reaction1.png

  • 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

File:Reaction2.png

  • 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)

File:ZhangLab 2 2011-10-11 11hr 06min-SeqLib-PC.jpg