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From 2011.igem.org

Steve, Dr Liz present

We picked single colonies from the pET-Taq (4A4) in the 10-alpha strain of E. Coli growing on one of the two plates Dr Burkett brought for us. Made 5 samples for incubation in the 37° shaker:

Each of the first 4 tubes contained 3ml of the liquid growth medium, while the fifth tube only had 1.5ml. We lightly touched 4 separate colonies, putting those into the first 4 tubes. We dropped the tip used to pluck each cell sample into its respective tube. For the 5th tube we again touched the same colony that we touched for Tube 4. We are curious to see if there is a difference in growth between samples 4 & 5. Our media was LB enhanced with 50mM d-glucose (also called glucose or dextrose)..

We refreshed our ROOM-Temp TSA plus AMP plates (given to us by Cheryl Brown) with more AMP. We used 100ul on each plate, spreading with an ethanol-dipped and flamed glass wand. They absorbed the liquid sitting gel-side-down for just under an hour credit we taped them back into their plastic sleeve, gel on too, and put in our box in the cold room.

We set up 4 positive control PCR rxs using most of the remaining sample of purified pET-Taq (4A4) as template. We created a tube of 0.06 ug/ul template by mixing the full-strength pET-Taq with the 4A4 that had been diluted to a 1/100 concentration. The two variables were template concentration and annealing temp. As we used the Pol1 with BioBrick-tails for primers we followed the protocol suggested by the polymerase manufacturer and took the Tm of each primer (60.9 and 59, respectively) , decided to use their average, 60°C, and subtracted 5° as per manufacturer instructions. We label the tubes to be PCR'd with this 55° annealing temp with "A". Because Dr Liz has an idea the primers with tails may avtually have Tm's lower than those found using standard online primer-design software, we labeled tubes to be PCR'd with a 53°C annealing temp with "B".

So machine settings were Machine A Machine B 95 C for 15 min 95 for 15 min 35 cycles of 35 cycles of

    94 C for 1 min                                       94 C for 1 min
    55 C for 1 min                                       53 C for 1 min
    72 C for 7 min                                       72 C for 7 min

(cycling ends) (cycling ends) 72 C for 10 min 72 C for 10 min

The other PCR variable, template concentration was this: Tubes with 0.5 ug of template were labeled "1" and those with 0.25ug of template "2". We also put the letter M in each of the 4 labels, to distinguish these samples from samples we already have made and used.

We used Qiagen's HotStar Polymerase Master Mix, which had the polymerase, dNTPs and buffer. We brought the samples up to 95°C in the PCR machines before adding the Master Mix.

More tomorrow.