Jeff:LabNotes/Microbiome/2009-9-23: Difference between revisions

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(New page: == '''Extraction of 500 bp plasmid insertion''' == *The biomiga miniprep II kit was used for extraction *The product was run on a 1 % agarose gel for 40 min *No restriction was used yet s...)
 
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[[Image:9-23-09 gel.jpeg|400px]]
[[Image:9-23-09 gel.jpeg|400px]]
== '''MDA in an Agarose base''' ==
''Try a new technique for MDA by keeping agarose warm and adding last.''
'''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}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
|-
| H20||0||0||0||0||0||0||0
|-
| .75% LMT Agarose||12.6||12.1||11.6||11.1||10.6|
| C12 bound to bead||0||0.5||1||1.5||2|
| N6 free primer||3||3||3||3||3|
| Buffer||6||6||6||6||6|
| NTPs||2.4||2.4||2.4||2.4||2.4|
| SYBR Green 2X||3||3||3||3||3|
| Phi 29 Enzyme||3||3||3||3||3|
| Total||30||30||30||30||30|
|
|}
*Add all reagents to master mix except for agarose and enzyme
*Keep agarose warmed and in its liquid phase.  After all reagents are added besides the enzyme, add the agarose
*Heat up bacteria at 90 C for 2 min to lyse. 
*While heating up, add Enzyme to master mix
*Cool bacteria at 40 C for 2 min and then put on ice
*While cooling at 40 C,  mix thouroughly
*Add 10 uL of bacteria in agarose to 8 tubes  and 10 uL .75% agarose to 2 tubes (NTC).  Put tubes on ice. 
*Add 10 uL of master mix to each respective tube.  Final conc. of agarose is .375%
*Perform real time MDA with the opticon machine
'''Results'''
The results show that the new method did not work.  The agarose was probably too hot when added to the master mix and thus killed the enzyme.  Furthermore, even though it was kept hot longer, it was still sticky and thus difficult to work with.  I believe that we will have to find a better concentration of agarose to work with before we can continue because the MDA is too inconsistent.
[[Image:9-23-09 mda.jpeg|400px]]

Latest revision as of 17:35, 24 September 2009

Extraction of 500 bp plasmid insertion[edit]

  • The biomiga miniprep II kit was used for extraction
  • The product was run on a 1 % agarose gel for 40 min
  • No restriction was used yet since it needs to be ordered

Results

The gel shows a similar band as the previous gel. Thus, a restriction enzyme must be used in order for the DNA to properly move through the gel.

File:9-23-09 gel.jpeg


MDA in an Agarose base[edit]

Try a new technique for MDA by keeping agarose warm and adding last.


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 C12 bound to bead 0 0.5 1 1.5 N6 free primer 3 3 3 3 Buffer 6 6 6 6 NTPs 2.4 2.4 2.4 2.4 SYBR Green 2X 3 3 3 3 Phi 29 Enzyme 3 3 3 3 Total 30 30 30 30
  • Add all reagents to master mix except for agarose and enzyme
  • Keep agarose warmed and in its liquid phase. After all reagents are added besides the enzyme, add the agarose
  • Heat up bacteria at 90 C for 2 min to lyse.
  • While heating up, add Enzyme to master mix
  • Cool bacteria at 40 C for 2 min and then put on ice
  • While cooling at 40 C, mix thouroughly
  • Add 10 uL of bacteria in agarose to 8 tubes and 10 uL .75% agarose to 2 tubes (NTC). Put tubes on ice.
  • Add 10 uL of master mix to each respective tube. Final conc. of agarose is .375%
  • Perform real time MDA with the opticon machine

Results

The results show that the new method did not work. The agarose was probably too hot when added to the master mix and thus killed the enzyme. Furthermore, even though it was kept hot longer, it was still sticky and thus difficult to work with. I believe that we will have to find a better concentration of agarose to work with before we can continue because the MDA is too inconsistent.

File:9-23-09 mda.jpeg