Freimoser:Protocols:SmashGrabYeastDNA: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
mNo edit summary
 
(3 intermediate revisions by the same user not shown)
Line 1: Line 1:
{{Template:FreimoserTopTemplate}}
{{Template:FreimoserTopTemplate2}}
<html>
<html>
<script src="http://www.google-analytics.com/urchin.js"
<script src="http://www.google-analytics.com/urchin.js"
Line 12: Line 12:


== Protocol: Smash & grab yeast DNA miniprep==
== Protocol: Smash & grab yeast DNA miniprep==
#Take about 1.5 ml of overnight culture (20-24h) from YPD medium. Add 0.3 g (roughly 0.3 ml) of glass beads, 0.2 ml of lysis buffer and 0.2 ml of a 1:1 mix of phenol and chloroform to the cell pellet in an 1.5 ml tube.
#Take about 1.5 ml of overnight culture (20-24h) from YPD medium. Spin cells down and remove supernatant.
#Add 0.3 g (roughly 0.3 ml) of glass beads, 0.2 ml of lysis buffer and 0.2 ml of a 1:1 mix of phenol and chloroform to the cell pellet in an 1.5 ml tube.
#Vortex the tube at top speed for 2 min.
#Vortex the tube at top speed for 2 min.
#Add 0.2 ml of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and vortex again for a few seconds.
#Add 0.2 ml of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and vortex again for a few seconds.
Line 21: Line 22:
# Discard the supernatant (use the aspirator; take care not to dislodge the pellet).
# Discard the supernatant (use the aspirator; take care not to dislodge the pellet).
# Rinse the pellet with 0.5 ml of cold, 70% ethanol, add the ethanol slowly down the side of the tube, then centrifuge for 3-5 sec.
# Rinse the pellet with 0.5 ml of cold, 70% ethanol, add the ethanol slowly down the side of the tube, then centrifuge for 3-5 sec.
# Remove the supernatant. Leave the tubes open and inverted for the pellets to dry (or dry the pellets under vacuum). Resuspend in 50 µl H2O.
# Remove the supernatant. Leave the tubes open and inverted for the pellets to dry (or dry the pellets under vacuum). Resuspend in 50 µl H<sub>2</sub>O.




Line 29: Line 30:


=== Glass beads: ===
=== Glass beads: ===
* Use 425-600 micron beads. Sigma G-9268 works well for us. To prepare the beads:
* Use 425-600 micron beads. Sigma G-9268 works well. To prepare the beads:
**Pour the beads (1 kg bottle) into a 2 litre beaker.
**Pour the beads (1 kg bottle) into a 2 litre beaker.
**Fill the beaker with concentrated HCl until the beads are  fully submerged. Let stand in a fume hood for 15 min.
**Fill the beaker with concentrated HCl until the beads are  fully submerged. Let stand in a fume hood for 15 min.

Latest revision as of 02:03, 24 October 2006

<html><style type='text/css'> A:link { 

   text-decoration: underline;
   font-size: 10pt;
   font-style: normal;
   font-family: arial, trebuchet ms;
   color: #003399;

}

A:visited { 

   text-decoration: underline;
   font-size: 10pt;
   font-style: normal;
   font-family: arial, trebuchet ms;
   color: #003399;

} A:active { 

   text-decoration: none;
   background-color: #99ccff;
   color: #660099;

}

A:hover { 

   text-decoration: none;
   color: #ff0033;

}

.navbar{ 

 padding: 0px;
 width: "100%";
 font-family: Arial;
 font-size: 10pt;

} </style></html>

Florian's Lab    People     Research     Publications     Protocols     Recipes     Group info     Contact    

<html> <script src="http://www.google-analytics.com/urchin.js" type="text/javascript"> </script> <script type="text/javascript"> _uacct = "UA-564843-2"; urchinTracker(); </script> </html>


Protocol: Smash & grab yeast DNA miniprep

  1. Take about 1.5 ml of overnight culture (20-24h) from YPD medium. Spin cells down and remove supernatant.
  2. Add 0.3 g (roughly 0.3 ml) of glass beads, 0.2 ml of lysis buffer and 0.2 ml of a 1:1 mix of phenol and chloroform to the cell pellet in an 1.5 ml tube.
  3. Vortex the tube at top speed for 2 min.
  4. Add 0.2 ml of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and vortex again for a few seconds.
  5. Spin the tubes for 5 min (room temperature) at top speed in an microcentrifuge.
  6. Transfer the aqueous (upper) phase (0.38 ml) to a fresh tube, using a new pipette tip for each sample. Discard the tube with the glass beads.
  7. Add 2 volumes of 100% ethanol at room temperature. Mix thoroughly.
  8. Centrifuge for 2-3 min at room temperature.
  9. Discard the supernatant (use the aspirator; take care not to dislodge the pellet).
  10. Rinse the pellet with 0.5 ml of cold, 70% ethanol, add the ethanol slowly down the side of the tube, then centrifuge for 3-5 sec.
  11. Remove the supernatant. Leave the tubes open and inverted for the pellets to dry (or dry the pellets under vacuum). Resuspend in 50 µl H2O.


Material:

Glass beads:

  • Use 425-600 micron beads. Sigma G-9268 works well. To prepare the beads:
    • Pour the beads (1 kg bottle) into a 2 litre beaker.
    • Fill the beaker with concentrated HCl until the beads are fully submerged. Let stand in a fume hood for 15 min.
    • Wash with tap-distilled water until the pH is neutral. It helps to run the water through glass tubing jammed to the bottom of the beaker so that the water flows up from the bottom of the beaker.
    • Transfer the beads to a baking dish and bake (overnight is good).
    • Pour the beads into a sterile bottle.


Lysis buffer

  • 10 mM Tris, pH 8.0
  • 1 mM EDTA, pH 8.0
  • 100 mM NaCl
  • 1% SDS
  • 2% Triton X-100


Source

Based on M. D. Rose, F. Winston, and P. Hieter (1990): Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Freimoser:Protocols:SmashGrabYeastDNA&oldid=81442"