Jeff:LabNotes/Microbiome/2009-7-9

From ZhangLabWiki
Jump to navigation Jump to search

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.