Kun:LabNotes/ExonomeSeq/2008-1-24: Difference between revisions

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(New page: ==Exp.1 Library construction on the new capturing reaction started on 01/16/2008== ===Mme I digestion on the 2nd round PCR amplicon=== 500bp+ 2nd PCR all size 2nd PCR ...)
 
 
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===USER enzyme digestion===
===USER enzyme digestion===
Add 2ul USER enzyme into 18ul of DNA in EB buffer, 37C 1h.
Add 2ul USER enzyme into 18ul of DNA in EB buffer, 37C 1h.
===End repair===
Set up end repair reaction (with NEB quick blunting kit)
                  USER       
      DNA        19ul       
      10X buffer 2.5ul       
      1mM dNTP  2.5ul       
      Enzyme mix  1ul         
      RT for 30min -> 75C 10min


===SolN6 primed synthesis==
===SolN6 primed synthesis===
                         SolF/Rn6     
                         SolF/Rn6     
       DNA                10ul       
       DNA                10ul       
Line 23: Line 31:


===Ligation===
===Ligation===
                        N6 primed       
                            USER     
       DNA                  10ul              
       DNA                  15ul              
   50uM Solexa_1            1ul              
   50uM Solexa_1            2ul              
   50uM Solexa_2            1ul                
   50uM Solexa_2            2ul                
   2X Quick ligation buffer 15ul                
   2X Quick ligation buffer 20ul                
   Quick ligase              1ul                
   Quick ligase              1ul
  H2O                      2ul               
   mix well -> room temperature for 20min -> 75C 10min
   mix well -> room temperature for 20min -> 75C 10min
   Purified with MinElute columns.
   Purified with MinElute columns.
Line 49: Line 56:
     50X SYBG I            0.2ul                      0.2ul
     50X SYBG I            0.2ul                      0.2ul
     98C 30sec -> 18 cycles of (98C 10sec -> 65C 20 sec -> 68C 30sec) -> 72C 3min ->15C hold
     98C 30sec -> 18 cycles of (98C 10sec -> 65C 20 sec -> 68C 30sec) -> 72C 3min ->15C hold
==Exp.2 Probe prep for Cosmic3760, CpG28k and CES27k==
PCR (with NEB Taq master mix)
                                    Cosmic3760              CpG28k            CES27k
                              x1          x25   
        Cosmic3760(20nM)    0.1ul      2.5ul  CpgCES (20nM) 2.5ul            2.5ul
        2x Taq master mix    50ul      1250ul              1250ul          1250ul
        100uM AP1V41U      0.4ul        10ul                10ul  pAP1V6U    10ul
        100uM pAP2V4        0.4ul        10ul                10ul  phosAP2V6  10ul
        H2O                49.3ul      1230ul              1230ul          1230ul   
94C 3min -> 20 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold
Ethanol precipitation.
Resuspended in 170ul dH2O, add 20ul 10X Lambda exo buffer, 10ul 5U/ul lambda exo, 37C 3h -> 90C 10min.
Purify with Qiaquick columns. Yield: Cosmic3760=53ng/ul x 80ul; Cpg28k=33ng/ulx80ul; CES27k=33ng/ul x 80ul.
Added 4ul USER enzyme, 37C 2h.
Add 90ul H2O, 20ul 10X DpnII buffer, 10ul DpnIIV4/5ul DpnIIV4/5ul RE-DpnIIs, 94C 5min -> slowly  cool down to 37C -> add 10ul Dpn II -> 37C 2h -> add 5ul USER -> 37C 2h -> 75C 10min -> 4C hold.
I took 100ul Cosmic3760 and 100ul CpG28k out after 2-hour digestion. Did phenol/chloroform extraction, then PAGE purification. The palettes were resuspended in 10ul phosphorylation mix (1X T4PNK buffer, 1mM ATP, 1U/ul T4 PNK), 37C 10min. Then diluted to 20ul with ddH2O.
Yield:
*Cosmic3760: 6.2ng/ul => 194nM x 20ul
*CpG: 3.1nM => 97nM x 20ul
The remaining digestions were left until the reactions were completed. I heated the reactions at 95C for ~10min. Then did another round of PAGE on 100ul reactions on 01/26/08.
Yield:
*CES27k: 6.8ng/ul => 209nM x 20ul
*Cosmic3760: 7.1ng/ul => 218nM x 20ul
*Cpg28k: 3.4ng/ul => 104nM x 20ul

Latest revision as of 00:53, 28 January 2008

Exp.1 Library construction on the new capturing reaction started on 01/16/2008[edit]

Mme I digestion on the 2nd round PCR amplicon[edit]

          500bp+ 2nd PCR       all size 2nd PCR
              50ul                   50ul
 32mM SAM    0.5ul                  0.5ul
 Mme I 2U/ul   4ul                    4ul
 37C 1h -> 75C 20min -> 4C hold.
 Purify with Bioneer columns, then PAGE size selection.
 Split the purified DNA into two tubes, one for USER digestion and the other for SolN6 primed synthesis.

USER enzyme digestion[edit]

Add 2ul USER enzyme into 18ul of DNA in EB buffer, 37C 1h.

End repair[edit]

Set up end repair reaction (with NEB quick blunting kit)

                 USER         
     DNA         19ul        
     10X buffer 2.5ul        
     1mM dNTP   2.5ul         
     Enzyme mix   1ul          
     RT for 30min -> 75C 10min

SolN6 primed synthesis[edit]

                       SolF/Rn6     
     DNA                 10ul      
     10X Klenow buffer    5ul     
     100uM SolFn6         1ul
     10mM dNTP            2ul      
     H2O                 30ul      

94C 2min -> 16C 1min -> add 1ul 20U/ul Klenow exo- -> 16C 10min -> 37C 10min -> Clean up the reaction with G25 column, then add 6 ul 10X buffer, 2ul dNTp, 1ul 100uM SolRn6-> 94C 1min -> 16C 1min -> add 1ul 20U/ul Klenow exo-, 1ul T4 DNA pol-> 16C 10min -> 37C 10min -> purified with MinElute columns, eluted with 10ul EB.

Ligation[edit]

                           USER      
      DNA                  15ul              
  50uM Solexa_1             2ul              
  50uM Solexa_2             2ul               
  2X Quick ligation buffer 20ul               
  Quick ligase              1ul
  mix well -> room temperature for 20min -> 75C 10min
  Purified with MinElute columns.

Nick-translation[edit]

  DNA                    15ul
  10X ThermoPol Buffer    2ul
  10mM dNTP             0.4ul
  1mg/ml BSA              2ul
  8U/ul Bst pol           1ul
  65C 25min -> place on ice

PCR[edit]

    Nick-translated DNA      5ul       SolN6 primed DNA  5ul
    2X iProof master mix    25ul                        25ul
    100uM Solexa_PCR_up   0.25ul                      0.25ul
    100uM Solexa_PCR_lo   0.25ul                      0.25ul
    H2O                   19.3ul                      19.3ul
    50X SYBG I             0.2ul                       0.2ul
    98C 30sec -> 18 cycles of (98C 10sec -> 65C 20 sec -> 68C 30sec) -> 72C 3min ->15C hold

Exp.2 Probe prep for Cosmic3760, CpG28k and CES27k[edit]

PCR (with NEB Taq master mix)

                                   Cosmic3760              CpG28k            CES27k
                             x1          x25     
        Cosmic3760(20nM)    0.1ul       2.5ul  CpgCES (20nM) 2.5ul            2.5ul
        2x Taq master mix    50ul      1250ul               1250ul           1250ul
        100uM AP1V41U       0.4ul        10ul                 10ul  pAP1V6U    10ul
        100uM pAP2V4        0.4ul        10ul                 10ul  phosAP2V6  10ul
        H2O                49.3ul      1230ul               1230ul           1230ul    

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

Ethanol precipitation.

Resuspended in 170ul dH2O, add 20ul 10X Lambda exo buffer, 10ul 5U/ul lambda exo, 37C 3h -> 90C 10min.

Purify with Qiaquick columns. Yield: Cosmic3760=53ng/ul x 80ul; Cpg28k=33ng/ulx80ul; CES27k=33ng/ul x 80ul.

Added 4ul USER enzyme, 37C 2h.

Add 90ul H2O, 20ul 10X DpnII buffer, 10ul DpnIIV4/5ul DpnIIV4/5ul RE-DpnIIs, 94C 5min -> slowly cool down to 37C -> add 10ul Dpn II -> 37C 2h -> add 5ul USER -> 37C 2h -> 75C 10min -> 4C hold.

I took 100ul Cosmic3760 and 100ul CpG28k out after 2-hour digestion. Did phenol/chloroform extraction, then PAGE purification. The palettes were resuspended in 10ul phosphorylation mix (1X T4PNK buffer, 1mM ATP, 1U/ul T4 PNK), 37C 10min. Then diluted to 20ul with ddH2O. Yield:

  • Cosmic3760: 6.2ng/ul => 194nM x 20ul
  • CpG: 3.1nM => 97nM x 20ul

The remaining digestions were left until the reactions were completed. I heated the reactions at 95C for ~10min. Then did another round of PAGE on 100ul reactions on 01/26/08.

Yield:

  • CES27k: 6.8ng/ul => 209nM x 20ul
  • Cosmic3760: 7.1ng/ul => 218nM x 20ul
  • Cpg28k: 3.4ng/ul => 104nM x 20ul