Kun:LabNotes/ASE/2007-12-6: Difference between revisions

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PCR (with iTaq)
PCR (with iTaq)
                               x1        x16x2  
                               x1        x16x2  
         template(100nM)    0.1ul    2x0.4ul    
         template(100nM)    0.1ul    2x0.8ul    
         10x ABI buffer      10ul      32ul
         10x ABI buffer      10ul      320ul
         25mM MgCl2            6ul      192ul
         25mM MgCl2            6ul      192ul
         10mM dNTP            2ul        64ul
         10mM dNTP            2ul        64ul
         100uM pAP1V6U      0.3ul      9.6ul
         100uM pAP1V6U      0.3ul      9.6ul
         100uM phosAP2V6    0.3ul      9.6ul
         100uM phosAP2V6    0.3ul      9.6ul
         iTaq                  1ul        32ul
         Platinium Taq        1ul        32ul
         H2O                80.8ul      2600ul
         H2O                80.8ul      2600ul
94C 3min -> 20 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold
94C 3min -> 20 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold
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Ethanol precipitation, resuspend in 135ul dH2O.
Ethanol precipitation, resuspend in 135ul dH2O.


Lambda exo digestion: add 15ul 10X lambda exo buffer, 4ul lambda exo, 37C 4h, 75C 15min, ethanol precipitation, resuspend in 100ul dH2O.
Lambda exo digestion: add 15ul 10X lambda exo buffer, 4ul lambda exo, 37C 24h, 75C 15min, purified with Qiaquick columns.


Dpn II & USER digestion: Add 13.5ul 10X Dpn II buffer, 5ul RE-DpnIIn2s guide oligo, 95C 10min, terminate the PCR program and let the reactions cool down in 15min, add 10ul Dpn II, 5ul USER, 37C 8h, 75C 10min.
      CES22k #4 12ng/ul x 80ul
      CES22k #5  6ng/ul x 80ul
 
<b>Since the yield is quite low, I decided to stop at this step. I will repeat the PCR, and combine these amplicons with the ones from the new reactions for digestion.</b>


==Exp.3 PAGE purification of the remaining digested CES22k probes from 11-13-07==
==Exp.3 PAGE purification of the remaining digested CES22k probes from 11-13-07==
    [[Image:2007-12-07-CES22k-PAGE-purification.png|500px]]
    [[Image:2007-12-07-CES22k-Probe-quant.png|400px]]
    Concentration:
    CES22k #2: 31nM
    CES22k #4: 177nM
    CES22k #5: 127nM

Latest revision as of 22:52, 9 December 2007

Exp.1 PCR on the pooled captured DNA[edit]

I still have some capturing reactions from 11-21-07, 11-26-07, 12-03-07 left. I decided to pool the reactions from the same templates togethers, and do PCR to make more Solexa sequencing library. The reason is that, based on the quantification experiment did yesterday, there are still not enough number of circularized molecules per target.

                              one reaction        8 reactions
          Template:           5~10ul                  each
          10x Buffer:          9.0ul                  72ul
          25mM MgCl2           5.4ul                43.2ul
          25mM dNTP            1.2ul                 9.6ul
          100uM AmpSolV6F2     0.4ul                 3.2ul
          100uM AmpSolV6R2     0.4ul                 3.2ul
          iTaq                   1ul                   8ul
          50X SYBG I           0.8ul                 6.4ul
          H2O                   72ul                 576ul

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

Exp.2 Probe prep. for the CES22k set on two new libraries[edit]

Emily resynthesized two libraries for the CES22k set.

  1. CES22k 01783910053 (received Nov. 2007, same sequences as the above library) 100nM
  2. CES22k 10783810008 (received Nov. 2007, contains Solexa amplification adaptors) 100nM

PCR (with iTaq)

                             x1        x16x2 
        template(100nM)     0.1ul     2x0.8ul    
        10x ABI buffer       10ul       320ul
        25mM MgCl2            6ul       192ul
        10mM dNTP             2ul        64ul
        100uM pAP1V6U       0.3ul       9.6ul
        100uM phosAP2V6     0.3ul       9.6ul
        Platinium Taq         1ul        32ul
        H2O                80.8ul      2600ul

94C 3min -> 20 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold

Ethanol precipitation, resuspend in 135ul dH2O.

Lambda exo digestion: add 15ul 10X lambda exo buffer, 4ul lambda exo, 37C 24h, 75C 15min, purified with Qiaquick columns.

     CES22k #4 12ng/ul x 80ul
     CES22k #5  6ng/ul x 80ul

Since the yield is quite low, I decided to stop at this step. I will repeat the PCR, and combine these amplicons with the ones from the new reactions for digestion.

Exp.3 PAGE purification of the remaining digested CES22k probes from 11-13-07[edit]

   File:2007-12-07-CES22k-PAGE-purification.png
   File:2007-12-07-CES22k-Probe-quant.png
   Concentration:
   CES22k #2: 31nM
   CES22k #4: 177nM
   CES22k #5: 127nM