Matt:LabNotes/2014-9-5

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Revision as of 22:56, 8 September 2014 by >Mzcai (Created page with "==qMDA to detect any DNA contamination in UHRR== * Gel Image showed possible DNA contamination in UHRR **Could explain why sequencing analysis of ...")
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qMDA to detect any DNA contamination in UHRR[edit]

  • 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[edit]

  • Samples
    • 1.3 ug/ul UHRR
    • gDNA (human?)
    • NTC (nuclease-free H2O)
' 1 2 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
Components 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
Components 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[edit]

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