calculate transformation efficiency for a plate of 80 colonies correct for the d

Question

calculate transformation efficiency for a plate of 80 colonies

correct for the dilution by multiplying the number of colonies seen on the plate by 10

If you do not know how much DNA was added to the competent cells for this plate, assume that 100ng of DNA were added.

NEB Protocol for Transforming Chemically Competent Cells 1. Thaw a tube of Competent E. coli cells on ice for 10 minutes. The instructor will get these out of the 80 degree freezer for you. You may be given two different kinds of E.coli and should follow the protocol below for each one. (IF you have a secondary plasmid, add 1-5 ul containing 1 pg-100 ng of your secondary plasmid DNA to the cell mixture (if unsure, use 3uL). BUT this year you DON'T.) 2. Add 2 uL of your primary plasmid DNA. Carefully flick the tube 4-5 times to mix cells and DNA. Do 3. Place the cell-DNA mixture on ice for 5-30 minutes (depending on how much time is left in the lab 4. Heat shock in the water bath in the fume hood at exactly 42°C for exactly 10 seconds for BL21 cells, not vortex. (After this step, label your secondary plasmid well and store at -20 degrees as directed.) period). Do not mix 30 seconds for 5-alpha cells, and 45 seconds for JM109 cells. Do not mix. 5. Place on ice for 5 minutes. Do not mix 6. Pipette 950 ul of room temperature SOC media into the mixture. 7. Place at 37°C for 60 minutes. Shake vigorously (225-250 rpm) in the shaker at the back of EH 131 making sure your tubes are labeled distinctly (This step can be reduced, see below) 8. Warm selection plates to 37°C 9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC: for each E.coli sample, place 100uL of SOC media in two tubes. Pipet 10uL of the incubated cell mixture into one tube, mix well, and transfer 10uL of this mixture into the second tube and mix The first tube is a 1:10 dilution and the second is a 1:100 dilution. (You may be directed to make different dilutions.) 10. Spread 100 ul of each dilution onto a selection plate and incubate 14-16 hours at 37°C, Alternatively, incubate at 30°C for 20-24 hours or at 25°C for 48 hours 11. When incubation is complete, remove the plates from the incubator and check for colonies. Colonies at the center have grown in the presence of antibiotic, but colonies near the edge may not experience high concentrations of antibiotic and may not have maintained the plasmid as a result. Take pictures of your plates so that you can see the colonies. Seal the edges of plates with colonies with parafilm, label well and store at 4 degrees. In discussion with your lab instructors, choose which plate will be used to express your protein.

Explanation / Answer

Answer:

Based on the given information as per the protocol:

Transformation Efficiency (TE) is calculated as: TE = Colonies/ µg/Dilution

TE = Colonies/µg/Dilution

correct for the dilution by multiplying the number of colonies seen on the plate by 10

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