Kun:LabNotes/ASE/2007-11-20: Difference between revisions

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{| border="1" cellpadding="5" cellspacing="0" align="center"
{| border="1" cellpadding="5" cellspacing="0" align="center"
|-
|-
| align="center" style="background:#f0f0f0;"|''' '''
| align="center" style="background:#f0f0f0;"|''' '''
| align="center" style="background:#f0f0f0;"|''' '''
| align="center" style="background:#f0f0f0;"|'''Template'''
| align="center" style="background:#f0f0f0;"|'''Template'''
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| align="center" style="background:#f0f0f0;"|'''H2O'''
| align="center" style="background:#f0f0f0;"|'''H2O'''
|-
|-
| GM20431 gDNA(347ng/ul)||2.3ul||1ul||5.4ul||0.3ul
|1||GM20431 gDNA(347ng/ul)||2.3ul||1ul||5.4ul||0.3ul
|-
|-
| GM20431 cDNA (121ng/ul)||0.4ul||1ul||5.4ul||2.2ul
|2||GM20431 cDNA (121ng/ul)||0.4ul||1ul||5.4ul||2.2ul
|-
|-
| GC1F cDNA(48ng/ul)||1ul||1ul||5.4ul||1.6ul
|3||GC1F cDNA(48ng/ul)||1ul||1ul||5.4ul||1.6ul
|-
|-
| GC1EP cDNA(52ng/ul)||1ul||1ul||5.4ul||1.6ul
|4||GC1EP cDNA(52ng/ul)||1ul||1ul||5.4ul||1.6ul
|-
|-
| Hues6 gDNA(113ng/ul)||2.6ul||1ul||5.4ul||0ul
|5||Hues6 gDNA(113ng/ul)||2.6ul||1ul||5.4ul||0ul
|-
|-
| Hues6 cDNA(155ng/ul)||0.3ul||1ul||5.4ul||2.3ul
|6||Hues6 cDNA(155ng/ul)||0.3ul||1ul||5.4ul||2.3ul
|-
|-
| H1 gDNA(222ng/ul)||2.6ul||1ul||5.4ul||0ul
|7||H1 gDNA(222ng/ul)||2.6ul||1ul||5.4ul||0ul
|-
|-
| H1 cDNA(35ng/ul)||1.4ul||1ul||5.4ul||1.2ul
|8||H1 cDNA(35ng/ul)||1.4ul||1ul||5.4ul||1.2ul
|-
|-
| H2 gDNA(200ng/ul)||2.6ul||1ul||5.4ul||0ul
|9||H2 gDNA(200ng/ul)||2.6ul||1ul||5.4ul||0ul
|-
|-
| H2 cDNA(48ng/ul)||1ul||1ul||5.4ul||1.6ul
|10||H2 cDNA(48ng/ul)||1ul||1ul||5.4ul||1.6ul
|-
|-
| BJ MDA DNA(576ng/ul) ||2.6ul||1ul||5.4ul||0ul
|11||BJ MDA DNA(576ng/ul) ||2.6ul||1ul||5.4ul||0ul
|-
|-
| BJ cDNA(18ng/ul)||2.6ul||1ul||5.4ul||0ul
|12||BJ cDNA(18ng/ul)||2.6ul||1ul||5.4ul||0ul
|}
|}


94C 5min->60C 18h-> add 0.5ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 100nM dNTP) -> 60C 4h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C 4h -> 37C 1min -> add 3ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.
94C 5min->60C 18h-> add 0.5ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 100nM dNTP) -> 60C 4h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C 4h -> 37C 1min -> add 1ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.


[11-22-2007] PCR
[11-22-2007] PCR
                                                one reaction      12 reactions
                              one reaction      12 reactions
           Template:                           5ul                     each
           Template:             5ul                 each
           10x Buffer:                     9.5ul                 123.5ul
           10x Buffer:         9.5ul               123.5ul
           25mM MgCl2                     6ul                       78ul
           25mM MgCl2             6ul                 78ul
           25mM dNTP                   1.2ul                   15.6ul
           25mM dNTP           1.2ul               15.6ul
           100uM AmpSolV6F2     0.4ul                     5.2ul
           100uM AmpSolV6F2     0.4ul                 5.2ul
           100uM AmpSolV6R2    0.4ul                     5.2ul
           100uM AmpSolV6R2    0.4ul                 5.2ul
           Platinium Taq                     1ul                       13ul
           Platinium Taq         1ul                 13ul
           50X SYBG I                     0.8ul                   10.4ul
           50X SYBG I           0.8ul               10.4ul
           H2O                               76.7ul                   997ul
           H2O                 76.7ul                 997ul


94C 3min -> 10 cycles of (94C 45sec -> 58C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold.
94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold.
 
    Added 3 more cycles for #3, 4, 6, 8, 10, 11, 12.
    [[Image:2007-11-24-CES22k-capture-PCR.png|500px]]
    Since the amplification curve for #1 came up much earlier than others, I repeat the PCR on 3ul #1 with 10 cycles at the second stage of amplification.

Latest revision as of 23:12, 24 November 2007

Exp. 1 Perform MDA on the gDNA from BJ Fibroblast[edit]

       Template DNA(6ng/ul)  20ul
       10X RepliPhi buffer  2.5ul
       1mM N6               2.5ul
       95C 3min -> chill on ice -> add the reaction mix:    
       10X RepliPhi buffer 8.5ul
       25mM dNTP             4ul
       1mM N6              2.5ul
       H2O                  49ul
       100mM DTT             5ul
       RepliPhi phi-29       5ul
       30C 3h -> 85C 10min
       Purify with Microcon YM-100 => 576ng/ul

Exp. 2 Perform CES22k eSNP capture on PGP and hybrid samples[edit]

Template 10x buffer CES22k probes (277nM) H2O
1 GM20431 gDNA(347ng/ul) 2.3ul 1ul 5.4ul 0.3ul
2 GM20431 cDNA (121ng/ul) 0.4ul 1ul 5.4ul 2.2ul
3 GC1F cDNA(48ng/ul) 1ul 1ul 5.4ul 1.6ul
4 GC1EP cDNA(52ng/ul) 1ul 1ul 5.4ul 1.6ul
5 Hues6 gDNA(113ng/ul) 2.6ul 1ul 5.4ul 0ul
6 Hues6 cDNA(155ng/ul) 0.3ul 1ul 5.4ul 2.3ul
7 H1 gDNA(222ng/ul) 2.6ul 1ul 5.4ul 0ul
8 H1 cDNA(35ng/ul) 1.4ul 1ul 5.4ul 1.2ul
9 H2 gDNA(200ng/ul) 2.6ul 1ul 5.4ul 0ul
10 H2 cDNA(48ng/ul) 1ul 1ul 5.4ul 1.6ul
11 BJ MDA DNA(576ng/ul) 2.6ul 1ul 5.4ul 0ul
12 BJ cDNA(18ng/ul) 2.6ul 1ul 5.4ul 0ul

94C 5min->60C 18h-> add 0.5ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 100nM dNTP) -> 60C 4h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C 4h -> 37C 1min -> add 1ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.

[11-22-2007] PCR

                              one reaction       12 reactions
          Template:              5ul                  each
          10x Buffer:          9.5ul               123.5ul
          25mM MgCl2             6ul                  78ul
          25mM dNTP            1.2ul                15.6ul
          100uM AmpSolV6F2     0.4ul                 5.2ul
          100uM AmpSolV6R2     0.4ul                 5.2ul
          Platinium Taq          1ul                  13ul
          50X SYBG I           0.8ul                10.4ul
          H2O                 76.7ul                 997ul

94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold.

   Added 3 more cycles for #3, 4, 6, 8, 10, 11, 12. 
   File:2007-11-24-CES22k-capture-PCR.png
   Since the amplification curve for #1 came up much earlier than others, I repeat the PCR on 3ul #1 with 10 cycles at the second stage of amplification.