Etchevers:Notebook/STRA6 in eye development/2009/04/29

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DNA purification and prep for first qPCR on Roche 480 Lightcycler

Place tubes with beads on magnetic rack, add 150 μL recovered supernatant to appropriate tubes that had been stored in the cold.

Transferred tube contents to 15 mL tubes. Rinse earlier tubes with 300 μL isopropanol, add to tubes, and then add 5 volumes (1.5 mL) QG buffer or equivalent from the Qiagen DNA column cleanup kit (“PE” EtOH-containing buffer for rinsing the column, “EB” buffer for elution).

Follow the instructions in the cleanup kit, using 2 x 30 μL “EB” Tris-containing buffer to elute the DNA at the end.

Nicolas tried to dose these on the Nanodrop. We’ve been working with the dose for the total input fraction as 68ng/uL but the wavelength absorbance profile is aberrant, with a large peak at 225 nm and descending from there. So I’m not sure how much I believe it, although it’s the right order of magnitude.

We had used 1/20th of the total chromatin to determine the total input, then used 12.24 μg DNA as total input per antibody, and the C15 yielded approximately 80 μg DNA.

Sadaf’s notebook on page 78 has the paste-in of the sonication profile, which is good, though the gel is not optimal. Fragments extend primarily from 150 to 700 bp. Cf. early April Nicolas' eye chromatin profile, much prettier and same result.

We mostly tested some of the primers I had ordered to meet a certain number of criteria:

C7 peaks from SHSY5Y neuroblastoma cell line, cf. 2009 paper, for which the nearest genes are also nearest genes in the mouse ES and mouse neural progenitors-derived-from-ES cell lines;

Within these human peaks, some of which are quite sizeable, using the Broad Institute or GIS-ChIP PET tracks on the UCSC genome browser (corresponding to GM12878, K562 and p493 [for MYC] or hES3 [for H3K4me3] cell lines), I chose ChIP-chip peaks that were positive within the H3K4 tracks, with an assortment of mono- di- or trimethylation, although sometimes there was H3K9 (ac or me1) or H3K27me3, which signify possible repression as I understand things. These had different names according to the ENCODE region from which they were derived. One was a H3K4-bound sequence from DLX5, just in case.

We diluted regular patient DNA that we luckily re-dosed on the spectrophotometer, by ¼ so as to have 2 Ct between each point, and five points. This gives an idea if the primers and conditions are optimal or not, the point being not to keep all but to get a general idea.

For the total input DNA, instead of using the 68 ng/μL dilution, used 2.5 ng/μL in each qPCR fraction so as to determine for 10 ng total.

Each well in the “gamme” gets 5 μL mix + 1 μL primers (F+R diluted to 10 μM) and 4 μL dilute DNA to make up to 10 μL. Make a mix for each primer set. Using Sigma’s 2x qPCR Sybr Green mix.

We tested first my first six pairs of primers plus two diluted primers that Nicolas had provided: GAPDH and a chromosome 12 control region (see PDF “chr12negcontrolpic” that was supposed to be a negative region without anything much in it by way of features), and around 100kb from nearest neighboring genes. But these apparently corresponded to mouse sequences and would explain why they did not work on the run we did on 4/30.

Sadaf then diluted the other primers I had chosen, this time for the most part within the region of chromosome 8 that was deleted in one of the CGH array patients without C7 mutations. [I made a schematic of these primer choices in Powerpoint for her just before departure on May 7th]. Also added a choice near FOXG1.

Sadaf recorded the melting curves in her notebook, pages 82 to 96. I will just import the Cts from the table, raw data that could be used to reconstitute if needed.

There were two groups of primers; those with a “poor” efficiency at 1.75-1.85, and those with an “acceptable” efficiency, at 1.94-2.04. The ones we rejected in the current conditions were:

Desert 2, SDC2, Chr8ctrl, DLX5, CCNE2, ENr231, PTDSS1.