Ajeffs:bioanalyzer

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(Buffer compatibility warning)
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==Buffer compatibility warning==
==Buffer compatibility 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:
+
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 your 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. I then diluted the samples 50-fold (to about 1ng/μL), which seemed to solve the problem of samples leaving the wells during the vortex step (although, 1ng/μL seems too dilute to visualise fragments that are distributed over several kb). An alternative would be to purify the reactions (e.g., with MinElute) prior to running on the Bioanalyzer – which would largely preserve the concentration but would hopefully remove compounds causing the samples to fly out of the wells." [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. I then diluted the samples 50-fold (to about 1ng/μL), which seemed to solve the problem of samples leaving the wells during the vortex step (although, 1ng/μL seems too dilute to visualise fragments that are distributed over several kb). An alternative would be to purify the reactions (e.g., with MinElute) prior to running on the Bioanalyzer – which would largely preserve the concentration but would hopefully remove compounds causing the samples to fly out of the wells." [http://http://anatomy.otago.ac.nz/staff/AndrewClarke/ Andrew Clarke], University of Otago.

Revision as of 21:22, 6 June 2011


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; we have used it for 2-3 times longer with no obvious loss in performance - be warned: you do so at your own risk).

Ladders

  • The RNA ladders are in 0.2 mL tubes in Box 100 of the right-hand upright -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.
  2. Check the white O-ring on the priming station is not dirty and/or clogged.

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 compatibility 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 your 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. I then diluted the samples 50-fold (to about 1ng/μL), which seemed to solve the problem of samples leaving the wells during the vortex step (although, 1ng/μL seems too dilute to visualise fragments that are distributed over several kb). An alternative would be to purify the reactions (e.g., with MinElute) prior to running on the Bioanalyzer – which would largely preserve the concentration but would hopefully remove compounds causing the samples to fly out of the wells." Andrew Clarke, University of Otago.

After the run

Remove chip immediately.

Washing

After a chip run, perform the following procedure to ensure that the electrodes are clean (no residues are left over from the previous assay):

  1. Slowly fill one of the wells of the electrode cleaner with 350 uL nuclease-free water, then close the lid for 10 sec. (30 sec. for Pico).
  2. Remove the electrode cleaner and leave the lid up to air-dry the electrodes for 10 sec. (30 sec. for Pico).
  3. For single chip runs, discard water and pipette fresh before each use, or if doing multiple chips, empty and refill the electrode cleaner every five chips.

Use a new electrode cleaner with each new kit. If unsure, check the assay-specific washing and contamination instructions from Agilent.

Decontamination

Wash with RNAse Zap according to the specific kit guide.

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