Jeff:LabNotes/Microbiome/2009-7-9
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Agarose in MDA[edit]
Test to see if filtering .75% LMT agarose removes artifacts for use in MDA
- Based on a recent paper, MDA was successfully performed in agarose.
- Want to see if LMT can be easily worked with in MDA
- Perform MDA in PCR tubes and compare to negative controls
Procedure
- Filter LMT agarose using 5 um pores
- Perform a serial dilution of bacteria in agarose to 1250 cell/uL
- Scale up the volume for MDA so that it 5x a normal reaction so the agarose is easier to work with
- The following volumes were used
Reagent 1 rxn 5 rxn 5x rxn NTC 5x rxn no enzyme H20 5.6 0 0 0 Primer (bound 2 10 10 10 to mag beads) Free primer (1mM) .5 2.5 2.5 2.5 Buffer 1 5 5 5 .75% LMT agarose 0 28 28 30.5 dNTP .4 2 2 2 SYBR Green 2X 0 0 0 0 Phi 29 enzyme .5 2.5 2.5 0 Template 0 0 0 0 Total 10 50 50 50 ---------------------------------------------------------------------------------------------------------
- Aliquot diluted e.coli into 2 tubes of 10 uL each
- Heat up 90 C for 2 min to denature
- For normal rxn, mix 10 uL of master mix with 10 uL of diluted e. coli
- For NTC, mix 10 uL of master mix with 10 uL agarose
- For no enzyme rxn, mix 10 uL of master mix with 10 ul of diluted ecoli
- MDA was performed in PCR tubes
- After MDA, heat up products at 99 C for 10 min
- Add 2 uL 50X SYBR Green to each tube
- Incubate at RT for 5 min
Results
The following were taken with 5X, FITC filter
File:7-9-09.jpeg MDA
File:7-9-09 ntc.jpeg Non template control
File:7-9-09 ne.jpeg No enzyme control
The results show that the filtering did work. The MDA showed great amplification of DNA. However, the NTC also showed much amplification, implicating that contamination occurred. For the no enzyme control, very little product was formed. Thus, MDA did work in agarose gel. It is essential to practice to avoid further contamination.