Ajeffs:bioanalyzer

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Buffer warning)
Line 23: Line 23:
===Buffer warning===
===Buffer warning===
-
Certain buffer components can alter surface tension characteristics and cause liquid to come out of the sample wells during vortexing, which will ruin your run in one way or another from contamination or current leaks. Most PCR and restriction digestion buffers are fine, but be wary if you have any detergents such as Triton X-100: look for liquid on top of the chip after the vortexing. If there is liquid either (i) stop; or (ii) dry the liquid and take a punt. Solutions to this issue are to (i) dilute the sample, although this may dilute you sample below a useful level; or (ii) clean the samples with a column. The following reagents have interfered with the vortexing step:
+
Certain buffer components can alter surface tension characteristics and cause liquid to come out of the sample wells during vortexing, which will ruin your run in one way or another from contamination or current leaks. Most PCR and restriction digestion buffers are fine, but be wary if you have any detergents such as Triton X-100: look for liquid on top of the chip after the vortexing. If there is liquid either (i) stop; or (ii) dry the liquid and take a punt. Solutions to this issue are to (i) dilute the sample, although this may dilute you sample below a useful level; or (ii) clean the samples with a column. We have found the following reagents have interfered with the vortexing step:
-
*"Running 1μL neat (~50ng/μL) aliquots of NEBNext dsDNA Fragmentase reactions. NEB do actually provide the composition of the reaction buffer, and it’s pretty standard – reactions contained 20mM Tris, 10mM MgCl2, 50mM NaCl, 0.15% Triton X-100. There was also BSA at 1mg/mL. My guess is that it’s the Triton X-100 that’s causing the problems (?). There’s also the buffer that the enzyme’s shipped in but there’s nothing unusual in that." [http://http://anatomy.otago.ac.nz/staff/AndrewClarke/ Andrew Clarke], University of Otago.  
+
*"Running 1μL neat (~50ng/μL) aliquots of NEBNext dsDNA Fragmentase reactions. NEB do actually provide the composition of the reaction buffer, and it’s pretty standard – reactions contained 20mM Tris, 10mM MgCl2, 50mM NaCl, 0.15% Triton X-100. There was also BSA at 1mg/mL. My guess is that it’s the Triton X-100 that’s causing the problems (?). There’s also the buffer that the enzyme’s shipped in but there’s nothing unusual in that." [http://http://anatomy.otago.ac.nz/staff/AndrewClarke/ Andrew Clarke], University of Otago.
===Decontamination===
===Decontamination===

Revision as of 18:37, 8 February 2011

Back to Eccles Lab > DGG Protocols

Contents

Bioanalyzer pre-flight check list

Reagents

  1. All reagents, except for the RNA ladders, are in the top door-shelf of the fridge in Hercus 210.
  2. Reagents, except ladder, must stand at room temperature for 30 minutes before use.
  3. Check the date on the filtered gel in the reagent box: discard and make up fresh if too old (Agilent say 4 weeks, but we have used it for 2-3 times longer with no obvious loss in performance).

Ladders

  • The RNA ladders are in 0.2 mL tubes in Box 153 of the NZORD -80dC in Hercus 125c. The ladders are heat-denatured and in 1 uL aliquots. Make sure you get the right ladder: Nano should be the top rack of two inside the box, Pico the second rack and on the bottom.
  • The DNA ladders are not stored at -20dC, and are in the box with the rest of the reagents at 4dC.

Hardware

  1. Check the settings on the priming station:
    • Position of the chip deck: A, B, C, or D.
    • Position of the syringe clip: top, middle, or bottom.

Tips

  1. Pipette gel and marker onto the bottom of the well, not the side.
  2. Follow the Agilent protocol exactly.
  3. Pipette 6 uL of marker into non-sample-containing wells during the first pass: this saves a second pass that adds 1 uL of extra marker to the first 5 uL of marker in non-sample-containing wells to make up the total volume to 6 uL.
  4. Always load sequentially from sample well number one.
  5. If running less than a full chip, reduce the run time by altering the number of samples to run on the "Instrument" page to match the actual number of samples you have, remembering that the sample order zig-zags from top-left to bottom-right.

Buffer warning

Certain buffer components can alter surface tension characteristics and cause liquid to come out of the sample wells during vortexing, which will ruin your run in one way or another from contamination or current leaks. Most PCR and restriction digestion buffers are fine, but be wary if you have any detergents such as Triton X-100: look for liquid on top of the chip after the vortexing. If there is liquid either (i) stop; or (ii) dry the liquid and take a punt. Solutions to this issue are to (i) dilute the sample, although this may dilute you sample below a useful level; or (ii) clean the samples with a column. We have found the following reagents have interfered with the vortexing step:

  • "Running 1μL neat (~50ng/μL) aliquots of NEBNext dsDNA Fragmentase reactions. NEB do actually provide the composition of the reaction buffer, and it’s pretty standard – reactions contained 20mM Tris, 10mM MgCl2, 50mM NaCl, 0.15% Triton X-100. There was also BSA at 1mg/mL. My guess is that it’s the Triton X-100 that’s causing the problems (?). There’s also the buffer that the enzyme’s shipped in but there’s nothing unusual in that." Andrew Clarke, University of Otago.

Decontamination

  1. 350 uL RNase Zap, 1 min.
  2. 350 uL nuclease-free water, 10 sec.
  3. Air-dry, 10 sec.
Personal tools