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==qMDA to detect any DNA contamination in UHRR== *[[Matt:LabNotes/2014-6-18 | Gel Image]] showed possible DNA contamination in UHRR **Could explain why sequencing analysis of RNA-seq has negligible rRNA despite using random hexamer RT primers *qMDA will amplify any DNA allowing rough quantification of DNA in the UHRR sample ===Procedure=== *Samples **1.3 ug/ul UHRR **gDNA (human?) **NTC (nuclease-free H2O) {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''1''' | align="center" style="background:#f0f0f0;"|'''2''' | align="center" style="background:#f0f0f0;"|'''3''' |- | A||1.25ul UHRR||1ul UHRR||0.75ul UHRR |- | B||NTC||NTC||NTC |- | C||100 fg gDNA||100 fg gDNA||100 fg gDNA |- | D||1 pg gDNA||1 pg gDNA||1 pg gDNA |- | E||10 pg gDNA||10 pg gDNA||10 pg gDNA |- | F||100 pg gDNA||100 pg gDNA||100 pg gDNA |- | G||1 ng gDNA||1 ng gDNA||1 ng gDNA |- | H||10 ng gDNA||10 ng gDNA||10 ng gDNA |} *Add 4.5ul ALS to each nucleotide sample (~1.25ul) on ice block for 10min {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||82 |- | KOH||8 |- | DTT||10 |- | EDTA||2 |- | Total||102 |} *Add 4.5ul NS (from Andrew's -20C MDA box) to each sample on ice block *Immediatey add 11.2ul MDA Master Mix on ice block {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Phi29 Buffer||2.3 |- | 1mM *N6||1 |- | 25mM dNTP||1 |- | 50X SYBR Green||0.1 |- | Phi29 Polymerase||1.8 |- | H2O||5 |- | Total||11.2 |} *Put in qPCR machine (preheated lid)and immediately start program **99 cycles of 6min @30C followed by a quantification of fluorescence ===Result=== [[File:QMDA_UHRR.JPG]] *The UHRR sample begins to increase in fluorescence between the cycles when 1ng and 10ng increase *The cycle at which the UHRR sample begins to increase matches the order of sample volume (1.25ul -> 1.00ul -> 0.75ul) *The DNA standards behaved as expected *Can confidently conclude there is ~5ng/ul of DNA in UHRR sample
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