diluted cells too much? help - (Dec/16/2006 )
I was running an experiment and think my calculations may be off. For anyone that has done Arabidopsis transformation, please tell me if my transformation will be ok.
I grew up an Agro colony with a mutant insert in LB + KAN selective media. Then, I made a small culture from a single colony in LB + KAN selective media. That took 3 days to get this small culture dense, but it looked good in the end. I transferred this into a bigger LB media culture and grew that up overnight. The culture got dense in a day & looked well, so it was ready to be prepared for Agro transformation. I centrifuged the broth to get my cell pellet and measured the OD on a spectrophotometer to what protocol said by resuspending pellet in 5% sucrose. The final volume of this resuspension came to be 600ml to reach the required protocol OD. So, that seemed to work out.
Now, at this point in the protocol, it says to add in Silwet L-77, a surfactant that aids in getting the bacteria into the plant. It says to use 0.05% of this but doesnt go into much depth. So, what I did was take 1000ml and measure out 0.5mg of Silwet L-77, put that into the 1000ml, and put this into my 600ml. So this came to be a 1600ml of my final mix.
I realize my calculation blunder here because I put in so much water into my 600ml. I should've measured out 0.5g in 1000ml H2O to equal 0.05% Silwet. The protocol never states how much Silwet to actually put into my resuspended mix and I already dipped some plants. Will they transform? The Silwet may not act most likely, but I'm mostly concerned because I had diluted my 600ml with so much H2O. Will this affect anything? Everything else looked fine before this.
It's not a hard question. Basically, I just want to know that because I added a lot of water in the end and not enough detergent, will my plants still undergo good transformation? this is for anyone who has done plant transformation.