Kun:LabNotes/ASE/2008-1-2: Difference between revisions

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Lambda exo digestion: to 120ul DNA, add 15ul 10X lambda exo buffer, 8ul lambda exo, 7ul H2O, 37C 4h, 75C 15min, add 10ul USER enzyme, 37C 2h. Ethanol precipitation, resuspended in 80ul ddH2O.
Lambda exo digestion: to 120ul DNA, add 15ul 10X lambda exo buffer, 8ul lambda exo, 7ul H2O, 37C 4h, 75C 15min, add 10ul USER enzyme, 37C 2h. Purified 110ul of #4 and 40ul of #5 with Bioneer column, very little DNA was recovered. Ethanol precipitation the remaining DNA, resuspended in 40ul (#4) and 125ul(#5) ddH2O.


Dpn II & USER digestion: Add 10ul 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 4h, 75C 10min.
Dpn II & USER digestion: Add 5ul/15 10X Dpn II buffer, 2ul/6ul RE-DpnIIn2s guide oligo, 95C 10min, terminate the PCR program and let the reactions cool down in 15min, add 4ul/10ul Dpn II, 2ul/8ul USER, 37C 4h, 75C 10min.


==Exp.3 Make gDNA-free cDNA from Hues29, Hues30, Hues33==
==Exp.3 Make gDNA-free cDNA from Hues29, Hues30, Hues33==

Revision as of 22:33, 4 January 2008

Exp.1 Capturing cSNPs with the eSNP84k probe set

Templates:

  1. NA20431, 347ng/ul, one reaction with the equal molar pool of all five probe sets, 800ng/reaction.
  2. GM20431 ds-cDNA, 122ng/ul, Lib #1: 1ng; Lib #2: 20ng; Lib #3 50ng; Lib #4: 100ng; Lib #5: 400ng
  3. GC1EP, 114ng/ul, Lib #1: 10ng; Lib #2: 20ng; Lib #3 50ng; Lib #4: 100ng; Lib #5: 400ng
NA20431 GM20431 GC1EP
Template 2.3 1(20ng) 1(20ng) 1(50ng) 0.85 3.4 1(20ng) 1(20ng) 1(50ng) 0.88 3.5
10x buffer 1.5 1 1 1 1 1 1 1 1 1 1
Lib #1 (384nM) 2 2 2
Lib #2 (580nM) 1.5 1.5 1.5
Lib #3 (331nM) 2.6 2.6 2.6
Lib #4 (500nM) 2.4 2.4 2.4
Lib #5 (460nM) 1.8 1.8 1.8
H2O 0.9 6 6.5 5.4 5.75 3.8 6 6.5 5.4 5.72 3.7

94C 3min->60C 40h-> add 1.5ul/1.0ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 1uM dNTP) -> 60C 1h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C hold -> 37C 1min -> add 2ul of exo mix (Exo I, Exo III, T7 exo)->37c 2 hour -> 94C 5min -> 4C hold.

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

PCR (with JumpStart Taq)

                             x1        x16x2 
        template(100nM)    0.05ul     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
        JumpStart Taq         1ul        32ul
        H2O                  80ul      2560ul

94C 3min -> 18 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold

Purify each amplicons with Bioneer 4 columns. Yield:

  • CES22k #4: 63ng/ul x 120ul
  • CES22k #5: 62ng/ul x 120ul


Lambda exo digestion: to 120ul DNA, add 15ul 10X lambda exo buffer, 8ul lambda exo, 7ul H2O, 37C 4h, 75C 15min, add 10ul USER enzyme, 37C 2h. Purified 110ul of #4 and 40ul of #5 with Bioneer column, very little DNA was recovered. Ethanol precipitation the remaining DNA, resuspended in 40ul (#4) and 125ul(#5) ddH2O.

Dpn II & USER digestion: Add 5ul/15 10X Dpn II buffer, 2ul/6ul RE-DpnIIn2s guide oligo, 95C 10min, terminate the PCR program and let the reactions cool down in 15min, add 4ul/10ul Dpn II, 2ul/8ul USER, 37C 4h, 75C 10min.

Exp.3 Make gDNA-free cDNA from Hues29, Hues30, Hues33

RNA clean-up

                    Hues29            Hues30          Hues33        
   total RNA       0.76ug/ul x14ul 0.79ng/ul x 14ul 0.8ng/ul x 12ul 
   10x buffer         3ul              3ul              3ul          
   H2O (DEPC)        17ul             10ul             0ul            
   DNase I(2u/ul)     2ul              2ul             2ul           
   RNase Inhibitor  0.5ul            0.5ul           0.5ul         
   37C 30min -> 75C 10min -> Cleanup with RNeasy columns

Yield:


First strand synthesis

                   Hues29   Hues30  Hues33
  RNA               25ul     25ul    25ul
  10mM dNTP          3ul      3ul     3ul
  50uM Oligo dT      3ul      3ul     3ul
  65C 5min -> place on ice for 1min
  Add
  10x RT buffer      6ul      6ul     6ul
  25mM MgCl2        12ul     12ul    12ul
  0.1M DTT           6ul      6ul     6ul
  RNase OUT(40U/ul)  2ul      2ul     2ul
  SS III RT(200U/ul) 3ul      3ul     1ul
  50C 50min -> 85C 5min

Second strand synthesis

Cleanup the first-strand reactions with G-25 columns.

  To the ~60ul reactions, add
  H2O (DEPC)       22ul      
  10ul NEBuffer 2  10ul
  10mM dNTP         3ul
  DNA Pol I(10U/ul) 4ul
  RNase H (2U/ul)   1ul
  Mix well, incubate at 16C for 2h, purified with MinElute columns.

Yield: Hues29: 11ng/ul x 15ul Hues30: 8ng/ul x 15ul Hues33: 8ng/ul x 15ul Not sure why the yield is so low. I lost a lot of RNA in the clean up step.