(r1) ProtocolsUVLibrary < Lab

---+ UV mutagenesis of Bacteria

---++ Determination of Optimal UV treatment

This procedure is used to determine optimal treatment which will be used for library generation. 

   1 Grow culture overnight as appropriate for strain.
   1 Pellet 1mL of overnight culture at 3k rcf for 5 minutes. 
      * see note 1
   1 Aspirate media.
   1 Resuspend in 1mL sterile saline.
   1 Label sterile petri dishes for each of the for each condition to be tested for each strain.
      * see note 2
      * Typical conditions (all in µJ/cm<sup>2</sup>):
         * 0 -- required for determining death rate!
         * 5,000
         * 10,000
         * 15,000
         * 20,000
         * 25,000
         * 30,000
   1 Near the UV crosslinker to minimize contamination concerns, transfer 120µl of cells to the center of each petri dish. Cells should form a distinct droplet. 
   1 Place the dish in the UV crosslinker for the appropriate treatment. Essential to remove the lid from the petri dish.
      * good practice to place the 0 sample in the crosslinker as well.
   1 Transfer 100µL from the droplet to a labeled tube some careful pipetting can increase number of cells recovered as cells may settle during experiment. Be careful not to introduce air bubbles.
   1 Plate for ~200 cells assuming 0 death rate.
      * replicate plating or higher number of cells can be used to increase confidence in death rate.
   1 Grow o/n
   1 Calculate death rate based on number of colonies.
      * see note 3
   1 Update following table for future reference.

---++ Table of previously determined optimal (~95-99% death ratios). Please update with additional results.
|*Strain ID*|* µJ/cm<sup>2</sup>*|*Strain description*|*Researcher*|
|SKO16|27,500|BWA25113 with plasmid|DED -- via SKO|
|unk|10,000|acenobacter|BR|


---++ Library Generation

   1 Grow culture overnight as appropriate for strain.
   1 Pellet 1mL of overnight culture at 3k rcf for 5 minutes in each of 4 eppendorf tubes.
   1 Aspirate media.
   1 Resuspend each in 1mL sterile saline.
   1 Label 2 sterile petri dishes with 0 and optimal µJ/cm<sup>2</sup> treatment for each strain.
   1 Near the UV crosslinker to minimize contamination concerns, transfer a total of at least 11 120µl droplets of cells distributed around the dish. Typically up to 6 droplets can easily be kept distinct on a single plate.
   1 Place each dish in the UV crosslinker for the appropriate treatment. Essential to remove the lid from the petri dish.
      * good practice to place the 0 sample in the crosslinker as well.
   1 Transfer 100µL from each droplet to a labeled tube. Some careful pipetting up and down can lead to recovery of more cells as they may settle during experiment. Take care to not introduce air bubbles.
   1 Pellet cells at 3k rcf for 5 minutes.
   1 Resuspend in appropriate growth media.
   1 Transfer appropriate amount of cells to o/n culture. This can either be a dilution or nearly the entire volume of cells depending on how large of a library you want to start with.
   1 Plate both treated and control for ~200 cells assuming 0 death rate to estimate total number of viable mutants.
      * replicate plating or higher number of cells can be used to increase confidence in death rate.
   1 Grow plate and culture o/n.
   1 Calculate number of mutants based on number of colonies.
      * This is expected to be very similar to previously calculated death ratios x the total number of cells treated.
   1 Freezedowns should be created for o/n culture of viable cells and possibly resuspended treated cells depending on downstream applications.

---+++ Notes

   1 Single attempt at using 1/3 of the total concentration of cells did not result in any noticable differences. DED SKO GFP project.
   1 Conceivably 1 petri dish could be used with multiple strains, but risk of contamination obviously higher. 
      * _This has not been attempted to my knowledge (remove if you attempt with confident results or warning to not attempt in future)._
   1 Not suggested to extrapolate kill ratios, best to test additional conditions if 95<sup>+</sup>% death rate not achieved.



   
-- Main.DanielDeatherage - 13 Feb 2014
Barrick Lab > ProtocolList > ProtocolsUVLibrary
Topic revision: r1 - 2014-02-13 - 17:37:11 - Main.DanielDeatherage