Kun:LabNotes/CpgSeq/2007-12-4: Difference between revisions

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(New page: ==Exp.1 PAGE purification of padlock oligos== I received the 36 CpGMIPv2 oligos. I pooled 20ul of the first 24 oligos into the tube CpGMIPv2L, and 20ul of the remaining 12 oligos into the ...)
 
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I made 1:10 dilution of the two mixtures (10ul into 90ul 1X TE), measure the concentration by Nanodrop.
I made 1:10 dilution of the two mixtures (10ul into 90ul 1X TE), measure the concentration by Nanodrop.
     CpGMIPv2L(10uM): 559ng/ul           2ul oligos + 10ul water + 12ul 2x Loading dye => 2ul per lane
     CpGMIPv2L(10uM): 559ng/ul   3ul oligos + 9ul water + 12ul 2x Loading dye => 2ul per lane
     CpGMIPv2S(10uM): 459ng/ul           1ul oligos + 5ul water + 6ul 2x loading dye => 2ul per lane.
     CpGMIPv2S(10uM): 459ng/ul   1.5ul oligos + 4.5ul water + 6ul 2x loading dye => 2ul per lane.
 
 
==Exp.2 Bisulfite conversion of DNA==
I decided to make a fresh batch of DNA since I have an impression that bisulfite converted DNAs are not stable.
Resuspend one vial of Bisulfite Mix in 800ul nuclease free water, votex for 5min. Set up reactions:
                                  RL          Jurkat
    DNA                          5ul x 2      5ul x 2
    Water                      15ul x 2      15ul x 2
    Bisulfite mix              85ul x 2      85ul x 2
    DNA Protection Buffer      35ul x 2      35ul x 2
Perform the bisulfite conversion on PTC-200 using the EpiTect program 99C5min->60C20min->99C5min->60C1h25min->99C5min->60C2h55min->20Chold.
 
Follow the EpiTect protocol to clean up the bisulphite converted DNAs.

Latest revision as of 01:50, 5 December 2007

Exp.1 PAGE purification of padlock oligos[edit]

I received the 36 CpGMIPv2 oligos. I pooled 20ul of the first 24 oligos into the tube CpGMIPv2L, and 20ul of the remaining 12 oligos into the tube CpGMIPv2S.

I made 1:10 dilution of the two mixtures (10ul into 90ul 1X TE), measure the concentration by Nanodrop.

    CpGMIPv2L(10uM): 559ng/ul   3ul oligos + 9ul water + 12ul 2x Loading dye => 2ul per lane
    CpGMIPv2S(10uM): 459ng/ul   1.5ul oligos + 4.5ul water + 6ul 2x loading dye => 2ul per lane.


Exp.2 Bisulfite conversion of DNA[edit]

I decided to make a fresh batch of DNA since I have an impression that bisulfite converted DNAs are not stable. Resuspend one vial of Bisulfite Mix in 800ul nuclease free water, votex for 5min. Set up reactions:

                                  RL          Jurkat
    DNA                          5ul x 2       5ul x 2
    Water                       15ul x 2      15ul x 2
    Bisulfite mix               85ul x 2      85ul x 2
    DNA Protection Buffer       35ul x 2      35ul x 2

Perform the bisulfite conversion on PTC-200 using the EpiTect program 99C5min->60C20min->99C5min->60C1h25min->99C5min->60C2h55min->20Chold.

Follow the EpiTect protocol to clean up the bisulphite converted DNAs.