Matt:LabNotes/2014-9-5
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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]
- 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