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.