TapeStation Library Check

Edit this page

The protocol for checking libraries on the TapeStation.

Background

The Agilent TapeStation provides an accurate, efficient quantification and quality assessment of RNA, DNA and protein samples. RIN numbers are given to RNA samples and quantification can be done on each individual band in your sample. Output comes as both a gel image and a size distribution electropharogram. Can handle strip tubes or plates.

Equipment & Reagents

  • 2200 TapeStation (Agilent Cat # G2965AA)

  • Loading Tips (1pk = 384 tips, Agilent Cat # 5067-5153) ** must use their tips

  • Optical Tube Strips (Agilent Cat # 401428) ** can use other tubes with exact same dimensions

  • Strip caps (Agilent Cat # 401425) ** can use other caps not needed for actual machine run

  • Kits:

    1. D1000

    • ScreenTape (Agilent Cat # 5067-5582)

    • Reagents (Agilent Cat # 5067-5583)

      1. High Sensitivity D1000

    • ScreenTape (Agilent Cat # 5067-5584)

    • Reagents (Agilent Cat # 5067-5585)

      1. D5000

    • ScreenTape (Agilent Cat # 5067-5588)

    • Reagents (Agilent Cat # 5067-5589)

      1. High Sensitivity D5000

    • ScreenTape (Agilent Cat # 5067-5592)

    • Reagents (Agilent Cat # 5067-5593)

Store all tapes in their box with the label facing towards you (Fig 1 below)

https://docs.google.com/drawings/d/1t0-f8TKZFptM4MrDHA_w4AtsOcy_YRR7boeD1aCk2mg/pub?w=1429&h=495

Important Notes READ BEFORE USING:

  • RNA Reagent kits and tapes will be GREEN and DNA Reagent kits and tapes will be BLUE double check before you get started

  • Make sure you use the correct tape and buffers for the size fragments that you are looking at:

  • Pairs only EVEN number of samples because the two probes move together. Account for one well for the ladder. If you have 2 samples + ladder, fill a 4th with buffer

  • Tapes can only do up to 16 samples at a time (2 columns of a 96 well plate or 2 strip tubes)

  • If you are doing a 96 well plate you need to replace the Tape after the 16 samples are complete. Computer program will notify you when this is okay to do.

  • NEVER OPEN TAPESTATION WHILE RUNNING SAMPLES unless you want to start over! This will throw out all your data because it completes calculations after it runs through every sample.

  • Always wear gloves when handling the tapes, oils on your hands can impact the accuracy of the reads

Things to do before starting

Protocol

Prep your samples

    • Only one well needs to be used for the ladder and it doesn’t matter which well you use just make sure to label properly in the spreadsheet
    • If the number of samples + 1 ladder is an odd number, you need to add an additional well with just buffer in the appropriate amount for the kit (e.g. D1000 4 uL of buffer). The machine only runs samples in twos.

Table 1 : Tapestation kits with the appropriate amount of sample buffer with either sample or ladder that should be mixed in each well

Kit Sample Buffer (uL) Sample or Ladder (uL) Total Volume (uL)
D1000 3 1 4
High Sensitivity D1000 2 2 4
D5000 10 1 11
High Sensitivity D5000 2 2 4
Genomic DNA 10 1 11
RNA 5 1 6
High Sensitivity RNA 1 2 3

    • Use the vortex next to the tapestation.
    • press START and it will vortex for a minute and then stop.

Run the Tapestation

    • If you are doing a plate, you will need to refill tips after each set of 16 samples completes. The program will alert you when it is time to refill them.

    • Usually only happens if the tape has had some lanes used before

https://docs.google.com/drawings/d/1ajGI6Yz1gpO-WCYh8056qCoPAlh_n4mYoNaOQg73TOc/pub?w=465&h=336

-   Tapes have a barcode that needs to be read by the machine. Look at the barcode inside the machine and match the direction to the barcode on the tape
-   If in the wrong orientation, the computer will spit back an error
-   Save the wrapping if you aren't using all the lanes to place tape back in for storage after run

    • Otherwise, a CSV file with names can be uploaded

https://docs.google.com/drawings/d/1joVrT3Hetfx3kbLPvPmTcUZlsE-SD004qC5AmqHDxq8/pub?w=239&h=283

    • Wait ~20 minutes per tape
    • Do not open lid until prompted- you will lose all your data and have to start over
    • The TapeStation does not show a time approximation

Download results

    • You can view the results as both a gel image and an electropherogram

    • All concentrations are outputted as a table below the electropherogram

    • Open the files that you wish to compare

    • NOTE you can only compare those DNA to DNA or RNA to RNA AND they must be from the same kit

    • You can make a report of all of your data by clicking ‘File Make a report’
    • Check off the items you want in the report and change the order you want them to appear by using the arrows