Inoculating 96-well plates for Nash assay

29 Jan 2015

Overview

Tips

• sterilize stuff in the hood before
• Be careful to never have the block upside down when inoculating or withdrawing fluid
• Be careful not to rotate your wrist and reverse the A-H order when moving fluid from one 96-well plate to another
• Avoid touching the top grid on the 96-well plate. We don’t want cells from one well to hop into an adjacent well. Think of this as keeping the doormat to a house clean so you don’t get dirt inside.

Things that can be done at “waiting steps”:

Prepare previous day(s):

• A plan of what you are trying to test
  • Just screening new random library variants?
  • Are there old colonies that need to be re-tested?
  • Do some samples need to be run with biological replicates?
• Plates with low enough colony density for picking colonies
• sterile toothpicks
• A 96-well plan with a color-coded cartoon of the pattern you are creating

Bring to the sterile hood:

• A printout of the exact pattern you plan to inoculate the plate with
• 96-well deep blocks
• reverse tweezers
• scissors
• multichannel 1mL pipette
• autoclaved toothpicks
• control and test cells
• media with antibiotics
• 1mL tips
• something sterile to put media in
• breathable sticker(s)
• sharpie(s)
• ethanol

First steps

• (optional) put everything except bacteria (and media?) in the UV hood and sterilize it by applying UV light
  • bacteria shouldn’t be put under UV light. I believe UV light penetrates the plastic we use
  • antibiotics in media might not withstand the UV light, so lets avoid exposing it.
• Label the plate with the date of inoculation and the plate number if multiple are being created
  • Use the H-row side of the block. Consistency will reduce errors.
• Label A-H on the right side of the block

Inoculate controls

• The high activity + control is inoculated on the diagonal running from A1 to H8. Some extra positions may be included.

Inoculate non-control wells

Wrapping up

• Note on your paper printout the wells that were busted. Examples include:
  ** inoculated with two different cell types
• Note in the gSpreadsheet which wells should be discarded from analysis.