Kun:LabNotes/ASE/2007-10-29: Difference between revisions

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*Starting from ethanol precipitated DNAs made at [[Kun:LabNotes/ASE/2007-10-18 |10/18/2007]].<br>
*Starting from ethanol precipitated DNAs made at [[Kun:LabNotes/ASE/2007-10-18 |10/18/2007]].<br>
*Pool two tubes of the same amplicons into ~100ul in total, add 11ul 10x Lambda Exo buffer, 4ul 5U/ul Lambda exonuclease. Incubate @ 37C for 2 hours, check 2ul of the products in both 8% TBE gels and 6% TBU gels.
*Pool two tubes of the same amplicons into ~100ul in total, add 11ul 10x Lambda Exo buffer, 4ul 5U/ul Lambda exonuclease. Incubate @ 37C for 2 hours, check 2ul of the products in both 8% TBE gels and 6% TBU gels.
                      8% TBE gel                            6% TBU gel
        [[image:2007-10-29-PCR-Lambda_exo-TBE.jpg]] [[image:2007-10-29-PCR-Lambda_exo-TBU.jpg]]
*I saw sharp bands in the non-denaturing gel, suggesting there are still a lot of dsDNA. I decided to incubate the reactions for another 4 hours. I probably used the wrong reverse primer for the eSNP84k sets. I should have used the phosAP2V4 instead of AP2V4, since lambda exonuclease doesn't cut without a 5'-phosphate group.
===Exp2: Repeat PCR amplification on the eSNP84k library===
Reactions eSNP84k set1-5 (100nM):
                              x 8 x 5
    Template        0.2ul      1.6ul x 5
    10X buffer      10ul      80ul x 5
    10mM dNTP        2ul      16ul x 5
    100uM AP1V41U  0.4ul      3.2ul x 5
    100uM phosAp2V4 0.4ul      3.2ul x 5
    AmpliTaq Gold    1ul      16ul x 5
    H2O              86ul      680ul x 5
    50X SYBR I      0.8ul      6.4ul x 5
95C 15min -> 17 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 4C hold
The curves came up a little bit later than the previous amplification using JumpStart Taq. I did 3 more cycles, and purified 200ul of reactions with QiaQuick columns. The yield was pretty low, so I added two more cycles and purified another 200ul reactions.
          Lib    20cyc      22cyc
          #1    10ng/ul    19ng/ul
          #2      8ng/ul    15ng/ul
          #3      7ng/ul    15ng/ul
          #4    10ng/ul    24ng/ul
          #5      6ng/ul    10ng/ul
I also purified the remaining reactions (400ul each) with ethanol precipitation.
Mix the DNAs of the same library, add 10ul NEBuffer 4, 2ul BciVI, incubate @ 37C for 2 hours, 65C 20min.
    [[image:2007-10-30-padlock_intermediate.jpg]]
Add 2ul T7 exonuclease, 37C 2h, add 2ul USER enzyme, 37C 2h.
For the ethanol precipitated DNAs, I resuspended them in 90ul dH2O, add 10ul 10x NEBuffer 4, 2ul BciVI, 37C 2h, 65C 20min, add 2ul T7 exo, 37C 2h.


        [[image:2007-10-29-PCR-Lambda_exo-TBE.jpg]] [[image:2007-10-29-PCR-Lambda_exo-TBU.jpg]]
    [[image:2007-10-31-eSNP84k-probe-prep.jpg|350px]]
 
For the batch I, add 3ul USER, 37C 2h. For the batch II, incubate @ 37C 2h, add 5ul USER, incubate @ 37C 4h. Check with PAGE gel.
 
Perform phenol extraction on the batch I, then concentrate with Vacufuge. <b> I lost all DNA in the vacufuge because I placed the rotor upside down and all liquid came out during spinning. I've turned the rotor over.</b>
 
    [[Image:2007-11-02-eSNP84k-probe-quant.png|400px]]


*I saw sharp bands in the non-denaturing gel, suggesting there are still a lot of dsDNA. I decided to incubate the reactions for another 4 hours.
    Estimated concentration:
    eSNP84k #1: 1ng/ul
    eSNP84k #2: 0.6ng/ul
    eSNP84k #3: 1.3ng/ul
    eSNP84k #4: 2ng/ul
    eSNP84k #5: 1.4ng/ul

Latest revision as of 05:39, 3 November 2007

Exp1: Probe prep (cont.)[edit]

  • Starting from ethanol precipitated DNAs made at 10/18/2007.
  • Pool two tubes of the same amplicons into ~100ul in total, add 11ul 10x Lambda Exo buffer, 4ul 5U/ul Lambda exonuclease. Incubate @ 37C for 2 hours, check 2ul of the products in both 8% TBE gels and 6% TBU gels.
                      8% TBE gel                            6% TBU gel
        File:2007-10-29-PCR-Lambda exo-TBE.jpg File:2007-10-29-PCR-Lambda exo-TBU.jpg
  • I saw sharp bands in the non-denaturing gel, suggesting there are still a lot of dsDNA. I decided to incubate the reactions for another 4 hours. I probably used the wrong reverse primer for the eSNP84k sets. I should have used the phosAP2V4 instead of AP2V4, since lambda exonuclease doesn't cut without a 5'-phosphate group.

Exp2: Repeat PCR amplification on the eSNP84k library[edit]

Reactions eSNP84k set1-5 (100nM):

                              x 8 x 5 
    Template        0.2ul      1.6ul x 5 
    10X buffer       10ul       80ul x 5 
    10mM dNTP         2ul       16ul x 5
    100uM AP1V41U   0.4ul      3.2ul x 5
    100uM phosAp2V4 0.4ul      3.2ul x 5
    AmpliTaq Gold     1ul       16ul x 5
    H2O              86ul      680ul x 5
    50X SYBR I      0.8ul      6.4ul x 5

95C 15min -> 17 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 4C hold

The curves came up a little bit later than the previous amplification using JumpStart Taq. I did 3 more cycles, and purified 200ul of reactions with QiaQuick columns. The yield was pretty low, so I added two more cycles and purified another 200ul reactions.

         Lib     20cyc       22cyc
         #1     10ng/ul     19ng/ul
         #2      8ng/ul     15ng/ul
         #3      7ng/ul     15ng/ul
         #4     10ng/ul     24ng/ul
         #5      6ng/ul     10ng/ul

I also purified the remaining reactions (400ul each) with ethanol precipitation.

Mix the DNAs of the same library, add 10ul NEBuffer 4, 2ul BciVI, incubate @ 37C for 2 hours, 65C 20min.

    File:2007-10-30-padlock intermediate.jpg 

Add 2ul T7 exonuclease, 37C 2h, add 2ul USER enzyme, 37C 2h.

For the ethanol precipitated DNAs, I resuspended them in 90ul dH2O, add 10ul 10x NEBuffer 4, 2ul BciVI, 37C 2h, 65C 20min, add 2ul T7 exo, 37C 2h.

    File:2007-10-31-eSNP84k-probe-prep.jpg

For the batch I, add 3ul USER, 37C 2h. For the batch II, incubate @ 37C 2h, add 5ul USER, incubate @ 37C 4h. Check with PAGE gel.

Perform phenol extraction on the batch I, then concentrate with Vacufuge. I lost all DNA in the vacufuge because I placed the rotor upside down and all liquid came out during spinning. I've turned the rotor over.

    File:2007-11-02-eSNP84k-probe-quant.png
    Estimated concentration:
    eSNP84k #1: 1ng/ul
    eSNP84k #2: 0.6ng/ul
    eSNP84k #3: 1.3ng/ul
    eSNP84k #4: 2ng/ul
    eSNP84k #5: 1.4ng/ul