Expression - Protein/Purification/Expression Day 1 ● The...

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Protein/Purification/Expression Day 1 10/2/19 The purpose of this lab was to purify the protein of interest . First step was to divide the plate into quads with a marker. Add 10 ml of DHFR “aqueous bacteria” to the plate in quad 1 Then, smear the DHFR with the inoculating needles onto quads 1-4 Day 2 10/7/19 We need to add 3 ml of LB ampicillin to a 50 ml tube & labeling the tube. 150ul of gulc to the 50 ml tube The bacteria and glucose was added into the tube and centrifuged for 18hrs Day 3 10/9/19 There will be an hr long of incubation for today’s lab. 100ul of our cells into 900ul of Lb amp solution, 1-10 dilution, aiming for .03 solution. We took 900ul of Lb amp solution into the cuvette and 100ul of our cells, then we will shake it. Now, we will place it into the incubator from the front to back. We measured the solution in the Genesis Spec 20 and it gave us a calculation of 0.114 of the absorbance. Since it less than 0.3, we will need to increase it by 0.3/(0.114)absorbance given*(10)*11= 2.9 of the overnight culture Now, we will be taking 2.9ul of our cells into a new tube. Next, we will be taking 8.9ul of the Lb Sterile Amp placing it into the new tube. After combining the two different solutions together, we will be placing our new tube into the shaking incubator for an hour. We took our new tube out with a remaining of 15 minutes left, so it’s been in the incubator for only 45 minutes. 100ul of sample, we will be labeling a small new tube with our “initials & un-induced” Adding 300ul of own sample & 600ul of Sterile Lb Amp solution into the cuvette Once we’ve done that, we will place the cuvette into the Genesis Spec 20. The new absorbance = 0.137 Our instructor added 25 “IPTG” into the induced sampler and we placed it into the heated shaking incubator Day 4 10/14/19 We don’t want too many bacteria because it will not express proteins, they will try to live. We will be vortexing the induced sampler Begin with 3 tubes, we will have 100ul of induced solution & the other will have 1.5ml of the solution into the two other tubes then placing them into the centrifuge. Aliquot is a subsection, we will be analyzing later. Taking one sample and putting it into another.
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Day 5 10/16/19 Today we will be separating soluble and adding insoluble proteins together.
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