June 12, 2006
Experiment KF1: Goal is to take some DNA from the MIT biobrick samples (listed below), to transform them so as to grow up a bunch more. Do a plasmid miniprep to isolate the DNA. Cut it the plasmid with the promoter with SpeI and PstI to creat a space for the GFP reporter to be ligated in. Thus the plasmid with the GFP in it will be cut with XbaI and PstI. Then we will run them out in a gel to seperate the fragments and select the two pieces we want. Then we will do a gel purification, a ligation, and ultimately a transformation. Hopefully what we end up with will have the same behavior as the E7104 plasmid in bacti - in other words, they should both express GFP and glow.
- Add DNA to TOP 10 competent cells from the -80 freezer.
- 20 minutes on ice.
- Heat shock in 42 degree water for 30 seconds (exactly).
- 2 minutes on ice.
- Add 200ul S.O.C. to each tube.
- Shake on the warm shaker 1 hr.
- Plate on carb plates and grow at 37 degrees o/n.
- Grow up colonies in LB next.
MIT samples to be transformed from the biobricks plate
- R0010 - lac operon promoter
- E7104 - T7 promoter and GFP
- E0241 - GFP
June 13, 2006
Growth of transformant colonies
- Fill a number of glass tubes (labeled with tape) with 5ml LB each.
- Add 50ul of ampcillin to each tube.
- Pick one colony with a sterile plastic pick and swirl it around in the LB. Repeat for each of the colonies you want with a different tube each time. (We did two colonies per plate, so 6 total because nothing grew on our negative control, as expected).
- Place in lager glass tubes in the warm shaker. Shake o/n.
- Do a plasmid miniprep next.
June 14, 2006
- lac operon promoter
- promoter and GFP
Qiagen Miniprep Protocol(from kit essentially)
- Remove growing LB cultures from warm shaker.
- Set aside 1mL of each sample for later glycerol storage at -80 if the transformation worked.
- Use an eppendorf tube to spin down the rest of the LB culture (probably in about 3 spins each - keep the pellet each time and remove the supernatant by just pouring it off. The final time, pipet the supernatant away so that there's nothing left but pellet.)
- Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 °C) and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. Pipet up and down. No cell clumps should be visible after resuspension of the pellet.
- Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
- Add 350 μl Buffer N3 and invert the tube immediately but gently 4–6 times. To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.
- Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
- Centrifuge for 30–60 s. Discard the flow-through. Spinning for 60 seconds produces good results.
- (Optional): Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
- Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 60 s.
- Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
- Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
- Use the nanodrop to determine the concentration of the DNA.
The nanodrop is used to measure the concentration of DNA in a tiny sample (1ul). The machine is located in the back of the larger lab.
- Turn on machine and computer.
- Clean silver platform top and bottom with ethanol on kimwipe.
- Click ND 1000 on the desktop to open the program.
- Load a water sample (1ul) directly onto bottom part of silver platform and press 0K on the computer to make sure it's clean.
- Wipe it off and load another sample of water to get the background. Press blank on the screen so that it calibrates.
- Wipe again. Load first sample and type in the Sample ID and press measure.
- Repeat this clean, load, measure process for each sample.
- Press show report to see all the results.
RE digest Protocol
- 8ul DNA
- 2.5 ul BSA
- 2.5 ul Buffer (look up which is optimal on neb.com)
- .5 ul Enz 1
- .5 ul Enz 2
- 11ul water
- Each reaction has DNA, BSA, Buffer, Enzymes, and water in an eppendorf tube and is allowed to incubate for 1 hour at 37 degrees.
- Inactivate the enzymes by placing tubes in the heat block at about 85 degrees for 15 minutes.
- In this case, we cut our R0010 samples (those with the lac operon promoter) with SpeI and PstI because we want this plasmid to be our backbone with the promoter already on it. Cutting here should allow the reporter to be ligated in right after the promoter.
- We cut E0241 samples (those with the GFP reporter) with XbaI and PstI to cut the reporter out of that plasmid.
- To prevent the vector from ligating to itself in the ligation step later, we need to remove any phosphates with CIP (calf intestinal phosphatase). The stock is 10,000 units/mL and we want 1 unit added to each backbone sample (in this case R0100). That means adding .1ul/rxn, which is very difficult, so we made up a dilution of 1ul CIP with 9ul water and each used 1 ul of it.
- Let those with CIP in them sit at 37 degrees for 1 hr.