Dinh/NOTES/2010-7-9

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DMR 220K Multivariate Capture Experiment[edit]

Purpose: To get fixed length capture for DMR220K probes set. Also, to optimize probe usage in capture.

  1. Annealing temperatures: 58C/60C
  2. dNTP concentration: 200uM vs 50uM
  3. Probes/Template ratio.

Template: Using bis-converted PGP1-Fib and PGP1-iPS

Probes Required:

22ul of DMR220K (21ng/ul, Kun 6-10-10)
12ul of CpG97KA (13ng/ul, Dinh 6-30-10)

Jie's CpG-Seq Program (no cycling), use PGP1-iPS as template[edit]

(JD) PGP1-iPS (40ng/ul) x 7ul = 280ng, 
         [dNTP, amt probes DMR 220K]
 58 C == [50uM, 44ng]-(B)
 60 C == [50uM, 44ng]-(D)
 (B and D) => 7ul template, 2ul probes, 1ul 10x Ampligase Buffer
(JD) Control: Jurkat(98ng/ul) x 3ul = 294ng,
          [dNTP, amt probes 97K]
 58 C == [50uM, 39ng]-(C)
 60 C == [200uM, 39ng]-(E)
(C and E ) => 3ul H2O, 3ul template, 3ul probes, 1ul 10x Ampligase Buffer

Kun's CpG-Seq Program, use PGP1-fib as template[edit]

(KZ) PGP1-fib (100ng/ul) x 3ul = 300ng, 
         [dNTP, amt probes DMR 220K]
 58 C == [200uM, 44ng]-(F), [50uM, 44ng]-(G), [200uM, 176ng]-(H)
 60 C == [200uM, 44ng]-(I), [50uM, 44ng]-(J), 
 (F,G,I and J ) => 3ul H2O, 3ul template, 3ul probes, 1ul 10x Ampligase Buffer
 (H) => 2.5ul H2O, 3ul template, 8ul probes, 1.5ul 10x Ampligase Buffer
(KZ) Control: Jurkat(98ng/ul) x 3ul = 294ng,
          [dNTP, amt probes 97K] 
 58 C == [200uM, 39ng]-(A)
 60 C == [50uM, 39ng]-(K)
 (A and K ) => 3ul H2O, 3ul template, 3ul probes, 1ul 10x Ampligase Buffer


Programs[edit]

KZ : cycle temperature decrease at -0.4C/cycle from 95C to 58/60C
JD : denature at 95C for 5 min then go to 58C/60C
Both: annealing at 58/60C for 36 hours, add 2ul SLN, incubate at 58/60C for 4 hours, add 2ul ExoI/III mix, incubate (1hr-JD)/(2hr-KZ), 94C for 5 min.

SLN Mix[edit]

               1x (200uM dNTP)     12x        1x (50uM dNTP)          14x
dNTP(1mM)          0.2             2.4            0.05                0.7
H2O                0.4             4.8            0.55                7.7
10xbuffer          0.1             1.2             0.1                1.4
Ampligase          0.1             1.2             0.1                1.4
Stoffel            0.2             2.4             0.2                2.8


Results[edit]

Amplified 10ul capture mix with Amp6.3F/R NH2 primers
B, C, D, E = 17 PCR cycles
A, F-K = 15 PCR cycles
File:MultivariateCaptureDMR220k-A.png File:MultivariateCaptureDMR220k-B.png


Comparing JD vs KZ: 97K control does not need the extra 2 cycles. PGP1-iPS capture seemed to require those two cycles because of low input concentration. => No noticeable differences between the two. 
97K probes set: performs better at 58C than 60C (C vs E), (A vs K)
220K probes set: capture is more efficient at 58C than 60C (F,H vs I,J). Results from 200uM and 50uM vary.
Low probes amount: capture amplicons has more smearing for high probes input (176ng). See F (44ng DMR220K) vs G (176ng DMR220K)

The following samples were purified and purified using 6% TBE gel for N2 adaptor protocol. B and D (PGP1-iPS) F,G, and H (PGP1-fib)

File:PGP1-ips, PGP1-f 220k capture lib quant.jpg
1. B-10.16ng/ulx20ul=203ng (PGP1-iPS captured at 58C, 44ng 220K probes (30:1), size selected)
3. D-12.76ng/ulx20ul=255ng (PGP1-iPS captured at 60C, 44ng 220K probes (30:1), size selected)
5. F-3.22ng/ulx20ul=64ng   (PGP1-fib captured at 58C, 44ng 220K probes (30:1), size selected)
6. G-1.18ng/ulx20ul=24ng   (PGP1-fib captured at 58C, 44ng 220K probes (30:1), size selected)
7. H-10.89ng/ulx20ul=327ng (PGP1-fib captured at 58C, 176ng 220K probes (100:1), size selected)
8. I-12.53ng/ulx30ul=376ng (PGP1-fib captured at 60C, 44ng 220K probes (30:1), non-ss)
9. J-4.28ng/ulx30ul=129ng  (PGP1-fib captured at 60C, 44ng 220K probes (30:1), non-ss)