Welcome to Benchling!

You're looking at a Protocol. Here are some things you can do with it:

Capture context, materials, and steps

  1. Document a list of materials.
  2. Add individual steps in the protocol.
  3. Add timers directly within your protocol.

Create protocol repositories for your lab

Insert tables with calculations

Attach protocols to Notebook entries

Create account

Sign up with GoogleI already have an account
Benchling is actively tested against the latest versions of Chrome, Firefox, Safari, and Edge. **It doesn't look like your current browser is supported** - for more information, [click here](/browsers).
Monospace normal
Monospace bold
0 of 0
Advanced Settings
    Leaf Vasculature, Sugar, and Starch Content.
    • Protocol
    • Relevant Items
    • Metadata
    Editing disabled on read-only protocol.

    Leaf Vasculature, Sugar, and Starch Content.

    Introduction

    Modified from the Ainsworth lab protocol. Designed by Steven D. Rowland and Amber Flores. Clearing of discs punched from tomato leaflets for vasculature tracing, sugar content, and starch content analysis.

    Materials

    • Stocks:
      • G6PDH – 100U/mL in 50uL Aliquots
      • Hexokinase – 1500U/mL
      • PGI – 700U/mL
      • Invertase – 300U/mg
      • Amyloglucosidase - >260U/ml
      • α-amylase - 850 FAU
    • Stock Reagents (Clearing)
      • 2mM HEPES in 80% EtOH.
    • Stock Reagents (Sugar and Starch Assay)
      • 10x Stock
        • 1M HEPES w/ ~63mM MgCl2. pH to 7.0 with KOH.
      • ATP & NADP
        • 100mM solution of each. Combine to make 50mM final concentration solution.
        • Make 1mL aliquots and store @ -20°C.
        • 1 aliquot per assay.
      • Sucrose Standards
        • 5mM to 0.08mM solutions 1/2X serial dilutions in 80% EtOH reagent.
        • These should be made fresh each assay. [100mM Sucrose can be stored in water @ -20°C as a stock and used to make standards in EtOH each time.]
      • G6PDH
        • Make 50uL aliquots of 100U/mL solution.
        • 1 aliquot per assay.
      • Starch Reagents
        • 0.5M Sodium Acetate Trihydrate pH 4.9. (3.402g in 50mL H2O)
        • 5M HCl
        • 5% NaOH
      • 50mM Sodium Acetate Buffer
        • Make a 10 fold dilution of the 0.5M Sodium acetate buffer in H2O.
        • 500µL per sample +1mL (per 20 samples)
      • Starch Degradation Mix (per 20 samples)
        • Spin 100µL of Amyloglucosidase and 100µL of α-Amylase @ 13k rpm for 5mins @ 4℃
        • Remove supernatent.
        • Resuspend in 0.5-1mL of 50mM Sodium Acetate Buffer.

      Procedure

      • Clearing for Vasculature
      1. Add 500µL 100% EtOH to 1.5mL Eppendorf tubes.
      1. Place leaf punches in to the tubes.
      1. Heat samples @ 80°C for 20 minutes.
      1. Discard the supernatent.
      1. Resuspend leaf in 500µL 100% EtOH, and repeat steps 3-4 as needed (3X).
      1. Resuspend leaf punch in 5% NaOH and heat @ 80°C for 5 minutes.
      1. Allow to cool for 15 minutes.
      1. Remove supernatent.
      1. Resuspend in 50% bleach for 30 mins @ RT.
      • Adjust time based on type of tissue (Chamber, Greenhouse, Field grown).
      • You can heat @ 80°C for 5 to 10 minutes if tough tissue.
      1. Rinse several times with water, then allow to sit in ddH20 to remove bubbles.
      1. Add 10 uL of 1% Safranin to each tube.
      • Check to make sure the Safranin doesn't precipitate out (this can happen if not all bleach is removed).
      • Let sit for at least 30 minutes, but longer is fine.
      1. Transfer discs to 50% Glycerol and allow to sit to remove excess Safranin in disc's.
      1. Place on slides to image.
      • Collection for Sugar and Starch Assays
      1. Add 500μL of 100% EtOH to 2mL Eppendorf tubes.
      1. Place leaf tissue punch (~3mg), weighed prior, to the tubes.
      1. Heat samples @ 80°C for 20 minutes.
      1. Remove the supernatent and transfer to 96 deep-well plate for storage (~80% of all sugar is extracted @ this stage).
      • Place in -20°C immediately if assaying at a later time.
      • It is critical that the supernatent be removed at this point and not at a later time as sugar can be lost.
      1. Resuspend leaf in 500μL of100% EtOH, Repeat step 13 (2X) discarding supernatent.
      • Alternatively, collect the supernatent as separate samples to check for residual sugars.
      1. Add 500μL 5% NaOH.
      1. Heat @ 80℃ for 20 mins.
      1. Cool @ RT.
      • Add 125μL 5M HCl and mix.
      1. Discard supernatent.
      1. Rinse w/ ddH20 2X.
      1. Resuspend in 500µL 50mM Sodium Acetate Buffer.
      1. Add beads
      • Bead beat (by hand) for 10 to 30 seconds.
      • The tube will fill with foam, this is normal.
      1. Spin for 1 mins @ 13K rpm.
      • If you wish to check for starch, add ~10µL IKI.
      • Set aside 50µL to load onto plate as negative control for residual sugars if desired.
      • For Potato tuber see section below.
      1. Add 25µL of Starch Degradation Mix
      • Final 1.3U/220U in reaction.
      1. Incubate at RT for 1 hour.
      1. Incubate @ 65°C for 23 hours.
      1. Inactivate enzyme by incubating @ 80°C for 5 mins.
      1. Store @ -20℃ until ready to run samples.
      • Potato Tuber
      1. From step 23 -> let sit for 1 to 24 hours.
      1. Spin @ 13k rpm for 5 mins.
      1. Collect supernatent and discard solid material.
      • Starch should be solubilized into the SAB supernatent.
      1. Proceed to step 24 - 28.
      • Day of Sugar Assay: (Volumes are for 192 reactions or 2 plates)
      1. Working Solution
      • 4mL of 10X Stock added to 36mL of ddH2O.
      1. Assay Buffer
      • 29mL of Working Solution mixed with 1mL ATP/NADP solution.
      1. Add 48uL of 1M MgCl2 to reaction.
      1. Add 1 aliquot of G6PDH to Assay Buffer.
      1. Invertase
      • Make 2mL (17.5mg) in Working Solution and keep on ice.
      1. Centrifuge 100U of Hexokinase @ 13000 rpm for 2 minutes @ 4°C. Discard supernatant. (67μL)
      • Add 2mL of Working Solution and keep on ice. Final 0.5U in reaction.
      1. Centrifuge 42U of PGI @ 13000 rpm for 2 minutes @ 4°C. Discard supernatant. (60μL)
      • Add 2mL of Working Solution and keep on ice. Final 0.21U in reaction.
      1. All of these can be made together in 6mL's of Working solution to skip the combination step later.
      • Assay
      1. Add 140uL of Assay Buffer to standards wells.
      1. Add 140uL of Assay Buffer to sample wells.
      1. Combine Hexokinase, PGI, and Invertase (for total sugar content measurement only).
      • Add 30μL of mix to to plate.
      1. Add 10uL of standards [18 Fold] to the first three columns
      1. Add 10uL of samples. [18 Fold]
      • Sugar and Starch separately.
      1. Incubate at RT overnight for reactions to stabilize. Read at 340nm on spec.

      Welcome to Benchling!

      Sign in to view data.

      Split Workspace