Jeff:LabNotes/Microbiome/2009-9-21: Difference between revisions

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== '''MDA in an Agarose base''' ==
''See what the optimum volume of beads is for MDA.''
'''Procedure'''
*Filter LMT agarose using 5 um pores
*Perform a serial dilution of bacteria in .75% LMT agarose to 1250 cell/uL.  Also dilute in water for + control.
*Scale up the volume for MDA so that it 3x a normal reaction so the agarose is easier to work with
*Since 10 uL of template will be used, no water will be used in the master mix to keep the concentrations constant
*Want to vary the volumes of beads to see which works best
*The following volumes were used
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Reagent'''

Revision as of 17:53, 22 September 2009

MDA in an Agarose base

See what the optimum volume of beads is for MDA.


Procedure

  • Filter LMT agarose using 5 um pores
  • Perform a serial dilution of bacteria in .75% LMT agarose to 1250 cell/uL. Also dilute in water for + control.
  • Scale up the volume for MDA so that it 3x a normal reaction so the agarose is easier to work with
  • Since 10 uL of template will be used, no water will be used in the master mix to keep the concentrations constant
  • Want to vary the volumes of beads to see which works best
  • The following volumes were used


Reagent ' ' ' ' ' ' '
H20 0 0 0 0 0 0 0
.75% LMT Agarose 12.6 12.1 11.6 11.1 10.6 10.1 9.6
C12 bound to bead 0 0.5 1 1.5 2 2.5 3
N6 free primer 3 3 3 3 3 3 3
Buffer 6 6 6 6 6 6 6
NTPs 2.4 2.4 2.4 2.4 2.4 2.4 2.4
SYBR Green 2X 3 3 3 3 3 3 3
Phi 29 Enzyme 3 3 3 3 3 3 3
Total 30 30 30 30 30 30 30