Bioanalyzer (Kathryn)

The bioanalyzer can be useful for quantifying and quality checking double stranded DNA, RNA, and proteins. This method of gel electrophoresis will tell you both fragment size and concentration for each fragment, using up only 1 uL of sample. The reagents and chips are expensive (for the High Sensivity chips, ~$100/11 samples/single use  chip), and have limited shelf life (4 months or less).

Rieseberg lab members have used this machine to check the quality of sequencing libraries and cDNA for microarray expression analyses. Various DNA and RNA kits are available, depending on what you want to measure, and their specs are at the bottom of this post.


A protocol I wrote for the HS DNA chip is here: Bioanalyze_DNA hs

The same protocol, with more pictures, but less crucial details, is here: Agilient HS DNA manual.

Here’s an example of cDNA samples I ran which came out ok: Successful HS DNA run

Protocols for other kits (other than HS DNA) differ in a few key areas: sample number, silver clip and base plate position on priming station, pressurization time, chip vortex speed, gel-dye matrix filter spin speed. Double check these things if running another kit.

Trouble shooting:

This machine can be very tricky. Some might even say… evil. If you are having problems, contact our sales rep from Agilent (Stephanie Howes <>) and the technical rep (Albert Grafsky <>) with the .xad file showing the problem. Be sure to tell them that the Rieseberg lab, and I in particualr, have had extensive dealings with them in the past, and that all future troubleshooting should take this into account (they are quite bad at tracking these things).

The somewhat useful Agilent troubleshooting guide is here: bioA troubleshooting guide

Things I have learned the hard way:

  1. Use only non-filter, NON-AUTOCLAVED tips when using the gel-dye matrix, or when loading sample. Apparently there is some kind of residue or debris associated with filter tips or autoclaved tips, which can block the channels on the chip and cause the entire run to fail. For this same reason, do not use gloves with powder.
  2. “Room temperature”, the temperature at which the reagents are suppose to equilbrate for 30 minutes before use is actually 25 C or higher. Our lab is typically 21 C. Sometimes the chip works anyway, but if you are having problems, try placing the reagents in a 30 C water bath or heat block for 30 mins before use.
  3. Do not use the bench centrifuge to filter spin the gel dye mix. Though it appears to run fine, it has caused problems in the past. Instead, use the centrifuge in the hood.
  4. Gel-dye mix is made and filtered in batches, typically 5 chips worth at a time. Though it appears that there is an excess of gel dye mix even after 5 chips have been run, do not use the remainder, as it is filled with ‘debris’. For this same reason, after filtering, do not, at any point, vortex the gel dye mix.
  5. Before loading a chip on the machine, double check for bubbles or liquid caught on the sides of the wells. This will cause the run to fail. Also check the back of the chip for fingerprints on the glass plate. Also check that you CANNOT see the etchings – if you can, that means a bubble has been pressurized into the etchings. These will cause the machine to fail.
  6. When pressurizing the chip, check that the white gasket bulges out over all sides of the well, and does not have any debris on it, or cracks in the rubber. You should feel pressure when pressing the syringe plunger down, and when the silver clip is released, the syringe plunger should rebound to at least the 0.3 mL mark. If the chip is not pressurized properly, the chip will fail.
  7. Salts in your sample buffer can cause the run to fail.
  8. RNAZap is required to clean the pins before running an RNA chip. However, this can leave a residue which causes the baseline of later DNA chips to be noisy. If this is a concern, run 2 – 3 RNAse free H2O chips before running your DNA chip, leaving each cleaning chip on the machine for up to 10 minutes.
  9. Make sure the dye (blue cap) and gel dye mix (funny shaped tube) are protected from light whenever possible. Light decay will cause the readings to be weak.
  10. Concentration can be tricky. The kits all have a specified sample concentration range at which they should run. However, this is on a per fragment basis. Therefore, if you expect to have multiple fragments in your sample, it can be hard to predict if your sample is neither too strong to run correctly, nor too weak to show up for each fragment. If you get optical errors, and the gel view for a sample is filled with black and white blotches, try diluting your sample, or using a less sensitive kit, such as the DNA 7500. One overly concentrated sample may cause the next few samples on the chip to fail as well.
  11. There is a diagnostics program on the machine, which involves two special chips. If you are having problems, go ahead and run it, but know that there are any number of ways in which your chip can fail, which will not be addressed by the diagnostics program.

Specifications – 2100 Bioanalyzer DNA Kits

Analytical Specifications DNA 1000 Kit DNA 7500 Kit DNA 12000 Kit High Sensitivity DNA Kit
Sizing range 25–1000 bp 100–7500 bp 100–12000 bp 50-7000 bp
Sizing resolution 25–100 bp: 5 bp
100–500 bp: 5%
500–1000 bp: 10%
100–1000 bp: 5%
1000–7500 bp: 15%
100–1000 bp: 5%
1000–12000 bp: 15 %
50-600 bp: ±10%
600-7000 bp:± 20%
Sizing accuracy ± 10% CV* ± 10% CV* ± 15% CV* ± 10% CV*
Sizing reproducibility 5% CV* 5% CV* 5% CV* 5% CV*
Quantitation accuracy 20% CV* 20% CV* 25% CV* 20% CV*
Quantitation reproducibility 25–500 bp:15% CV*
500-1000 bp:5% CV*
100-1000 bp:10% CV*
1000-7500 bp: 5 % CV*
100-1000 bp: 15% CV*
1000-12000 bp: 10% CV*
50-2000 bp: 15% CV*
2000-7000 bp: 10% CV*
Quantitative range 0.5-50 ng/µL* 0.5-50 ng/µL* 0.5-50 ng/µL* 5-500 pg/µL
Maximum salt concentration in sample 250 mM for KCl, 15 mM for MgCl2 250 mM NaCl 250 mM for KCl 15 mM for MgCl2 250 mM NaCl 250 mM for KCl 15 mM for MgCl2 250 mM NaCl 10 mM Tris and 1 mM EDTA
DNA 12/chip = 300 samples/kit
Physical Specifications DNA 1000 Kit DNA 7500 Kit DNA 12000 Kit High Sensitivity DNA Kit
Analysis time 35 minutes 30 minutes 30 minutes 45 minutes
Samples per chip 12 12 12 11
Sample volume 1 µL 1 µL 1 µL 1 µL
Kit stability 4 months (see box for storage temp.)
Kit size 25 chipsDNA 12/chip =
300 samples/kit
25 chipsDNA 12/chip =
300 samples/kit
25 chipsDNA 12/chip =
300 samples/kit
10 chipsDNA 11/chip =
110 samples/kit
* Respective DNA ladder as sample

Specifications – 2100 Bioanalyzer RNA Kits

RNA 6000 Nano total RNA Kit RNA 6000 Nano mRNA Kit RNA 6000 Pictotal RNA Kit RNA 6000 Pico mRNA Kit Small RNA Kit
Analytical Specifications
Quantitative range 25–500 ng/µL 25–250 ng/µL 50-2000 pg/µL of purified miRNA in water
Qualitative range 5–500 ng/µL 25–250 ng/µL 50–5000 pg/µL in water 250–5000 pg/µL in water 50-2000 pg/µL of purified miRNA in water
Sensitivity (S/N>3) 5 ng/µL in water 25 ng/µL in water 50 pg/µL in water or 200 pg/µL in TE 250 pg/µL in water or 500 pg/µL in TE 50 pg/µL in water**
Quantitation reproducibility 10% CV (within a chip) 10% CV (within a chip) 20% CV (within a chip) 20% CV (within a chip) 25% CV (within a chip)
Quantitation accuracy 20% CV* 20% CV* 30% CV* 30% CV*
Maximum sample buffer strength 100 mM Tris
0.1 mM EDTA
or 125 mM NaCl
or 15 mM MgCl2
100 mM Tris
0.1 mM EDTA
or 125 mM NaCl
or 15 mM MgCl2
50 mM Tris
0.1 mM EDTA
or 50 mM NaCl
or 15 mM MgCl2
50 mM Tris
0.1 mM EDTA
or 50 mM NaCl
or 15 mM MgCl2
10 mM Tris
0.1 mM EDTA
RNA 6000 Nano total RNA Kit RNA 6000 Nano mRNA Kit RNA 6000 Pico total RNA Kit RNA 6000 Pico mRNA Kit Small RNA Kit
Physical Specifications
Analysis time 30 minutes 30 minutes 30 minutes 30 minutes 30 minutes
Samples per chip 12 12 11 11 11
Sample volume 1 µL 1 µL 1 µL 1 µL 1 µL
Kit stability ≥4 months at 4°C ≥4 months at 4°C ≥4 months at 4°C ≥4 months at 4°C ≥4 months at 4°C
25 chips per kit RNA Nano 12/chip = 300 samples/kit RNA Pico 11/chip = 275 samples/kit

* Determined analyzing the RNA ladder as sample
** Measeured for the 40 nt fragment of the Small RNA

3 thoughts on “Bioanalyzer (Kathryn)

  1. Thanks a lot Kathryn for this comprehensive post.

    As someone who has only just started using the Bioanalyzer I’m going to report two simple errors that I have already made.

    1. I ran a chip and then went home without removing it from the machine. Fortunately, Kathryn caught this mistake and no harm was done. As Kathryn says – DO NOT leave chips in the machine. The electrodes need to be rinsed with a water chip at least a couple of times after each chip. And no chip, including the cleaning chips, should be left in the machine between runs.

    2. I primed a HS DNA chip – i.e. put 9ul of gel mix in the G well then lowered the syringe and pressed it down to its clip for 60 seconds – without properly clipping the priming station closed. That is, I lowered the top part, with the syringe, without clipping it into the lower half, with the chip. I ran this chip and it failed. The priming station does clip closed with an audible and palpable click – make sure you hear or feel it close properly before priming a chip.

  2. Me again.

    In September 2012 I ran 21 High Sensitivity DNA chips through the Bionanalyzer without a single failure!!!

    I cannot explain how I managed to run so many chips without a problem, I have certainly run failed chips in the past, however, I will note a couple of things that may have contributed.

    First, as I was running so many chips – 21 in a few days – I got the reagents out of the fridge in the morning and kept them out at room temperature all day. So, no into the fridge/out of the fridge cooling and warming of the reagents. Maybe that helped.

    Second, I managed to develop a fairly effective way to get air bubbles out of the gel-dye mix after aliquoting it into the gel wells of the chip. The key is to get the bubbles to the surface of the gel. This is not easy but two things help. First, time – for example, after you’ve primed the chip you aliquot 9ul of gel into the other three gel wells and then you aliquot the marker, the ladder and samples. If you don’t bother attacking bubbles in the three gel wells until after you’ve done the rest of the pipetting you will hopefully find that the bubbles have risen to the surface of the gell and are easy to pop. Basically, a few minutes is sometimes long enough for the bubbles to rise to the surface of the gel by themselves. Second – vigorous tapping on the side of the chip. Holding the chip, by its edges, very firmly you can pound on one side of it as hard as you can with a finger or tap it against something hard. You may have to do this for an annoyingly long time but it should bring bubbles to the surface of the gel.

    Once you have bubbles at the surface of the gel they will either spontaneously burst or you can poke them with a p10-20 pipette tip. If you poke a tip into the gel make sure you block the fat end of it with your finger. This will minimize the amount of gel that you remove by capillary action into the tip.

    Third, I was very thorough with the rinsing of the electrodes with water. I put 300ul or 350ul (whatever the correct volume is, I forget) of water into a cleaning chip and put it in the machine for 30 seconds or a few minutes several times before each run.

    Fourth, I was very careful with the chip priming. I watched the rubber seal compress onto the priming well closely to make sure it didn’t look asymmetrical or in any way abnormal. I changed the syringe at the beginning but not again – so it was the same syringe for all 21 chips.

    Good luck,

  3. One other thing.

    Please record usage on the reagent boxes!

    Each box of reagents contains two batches of gel that each get mixed with some dye, filtered and then used for five chips.

    Record the date of the gel preparation and each usage on the box and, for usage, on the tube. As Kathryn notes above there is a volume of gel/dye mix in each batch that is in excess and its not obvious to new users whether a tube is done or not without a record of the number of uses.


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