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Observation of the transformation from the night before (LovTAP-pMP)
Growth on all the plates (about 20 colonies on most plates and a single colony on the control plate).
Overnight culture
Protocol: Prepare Plasmid Extraction (culture for Miniprep)
- Select and number colonies on the plates.
- Prepare tubes of LB medium with the correct quantity of antibiotics (100 µg/ml for Amp, Spc or chloramphenicol).
- Amp can be found in the -20ºC freezer at Ecoli, labeled as "stock". It is 100 µg/µl, or 1000x.
- The tubes to be used are the 14 ml round bottom found in front of the iGEM drawers (Falcon). Culture with cap in the first step (loose) and close to the second step after culture.
- Touch each colony with a clean pipette tip and put it in a tube.
- Put in incubator.
Colonies from the plates were picked up and cultured for minipreps.
Miniprep
Protocol: Miniprep
The slim tubes can be centrifuged in the machine in front of the "Gel hood", at 4000 rpm for 10 min. The fatter ones, in the E. coli centrifuge by the fridge (the tip can be left inside, since it floats).
Pellets resuspended with RNase containing buffer (Resuspension Buffer R3, from Invitrogen, equivalent to Buffer P1 from Qiagen, in Sowmya's box in the fridge). Note: keep the buffer in ice if you are not bringing it back to the fridge for some minutes.
We then use the QIAGEN QIAprep Spin Miniprep Kit with their [http://www.qiagen.com/literature/render.aspx?id=370 protocol] (page 22) and a microcentrifuge.
All miniprep colonies of the ligations were used to extract DNA, except SEAP1 + LS (there wasn't a lot of growth, we used the 1:2 dilution).
Nanodrop of the minipreps
Protocol: DNA Concentration Measurement
- Take a 6 µl aliquote of the DNA and put back the main DNA tube in the fridge.
- Go to the room by the E.Coli lab (LBTM, not on Friday morning!) with:
- The 6 µl aliquote
- A 10 µl pipet
- Optionally, the buffer you used for DNA elution (there might be some next to the machine).
- The machine is the NanoDrop Spectrophotometer.
- On the computer, click on "Nucleic Acid".
- Put a 2 µl drop of (nuclease-free) water on the machine's tip as you are asked to and measure.
- Clean tips (both sides) with a quarter of tissue.
- Add 2 µl of the buffer you use and click on "Blank".
- Clean tips (both sides).
- Add 2 µl of your DNA sample and click "Measure".
- Clean tips (both sides) with a tissue.
- Take 2 measurements per sample (for averaging).
- Print the report when you are done
- Click on exit.
The important numbers are:
- 260/280 ratio, must be > 1.8
- 260/230 ratio, must be > 2 (too big, > 2.5? , might mean too much salts)
- Of course the DNA concentration.
The Nanodrop gave acceptable concentrations for all of the minipreps, between 200 and 400 ng/µl.
Digestion of LovTAP and pGL
Protocol: Restriction site digestion
- Look for the best pair of restriction sites, ideally with similar digestion temperatures and times.
- [http://tools.neb.com/NEBcutter2/ NEBcutter] for finding cutting enzymes.
- [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp Double Digest Finder] for the parameters.
- Calculate the amounts required of:
- DNA
- Buffer (usually from 10x to 1x)
- BSA, if needed (usually from 100x to 1x)
- Enzymes (depends on the amount of DNA)
- Water
- Get the recommended buffer (and BSA if needed) from the freezer and let defreeze.
- Mix all the ingredients, except DNA, in a tube.
- Note: Enzymes should stay no longer than a couple of minutes out of the freezer. Don't touch the bottom of the tubes! Don't vortex!
- Distribute the mix in as many tubes as DNA samples and add the DNA.
- Keep in the Thermomixer at the recommended temperature.
Sowmya's recommended amounts (50 µl total solution):
- 5 µl of 10x buffer
- 0.5 µl of 100x BSA
- 1 µl of each enzyme
- 5 µl of DNA
- 37.5 (up to 50 µl) of water.
Protocol based on what was done on July the 4th.
Protocol used for the Master mix: - 5 µl Buffer N4 10x - 5 µl BSA 10x - 1 µl XbaI (20 u/µl) - 5 µl DNA - 34 µl H20
- Comments
Re-verification of excised LovTAP & pGL fragments with a single digestion. Digestion of pGL backbone with NotI fragments generated 2922 and 1342 bp using small quantities of DNA.
Quantities in each tube:
- 5 µl buffer N2 10x - 5 µl BSA 10x - 5 µl DNA (pGL backbone) - 1µl Not1 - 34 µl H20
Quantities in each tube:
- 5µl buffer N2 10x - 5µl BSA 10x - 1µl Xba1 - 5µl DNA (excised LovTAP) - 34µl H20
Production of a new TAE solution
Protocol: TAE
To make 200 ml of %0x TAE :
- Tris base: 78.4 g
- Acetic Acid (100%): 11.4 ml
- EDTA: 20ml 0.5 M
- 292.25M --> 146.12 = 0.5 mol --> 1.46 g in 10 ml is 0.5M
- Comments
The TAE in the lab got sticky and contained unknown particles in it, so it was useful to make a new mix.
PCR and Gel Electrophoresis
Protocol: PCR
PCR is a reaction that makes it possible (and relatively easy) to amplify
a certain region of DNA. The first step is the selection of that region
(and the design of the relevant primers). Primer design can be done by hand, or by
using our Primer Design Helper. Once
done, order the primers (in our case, we ordered from them [http://www.idtdna.com/ IDT]).
When you've received the primers, prepare them and make sure you've got your PCR kit (we used the "Phusion® High-Fidelity DNA Polymerase"). Start preparing your master mix, the composition for one tube is:
1X Mastermix 20μl reaction, add in this order
| Reagent | Volume [μl] |
|---|---|
| Water | Complete to total volume of 20μl |
| HF-Buffer (5x) | 4 |
| DMSO (optional) | 0.6 |
| dNTPs | 0.4 |
| Forward primer (50μM) | 0.2 |
| Reverse primer (50μM) | 0.2 |
| Template (10ng/μl) | 0.5 |
| Phusion HF polymerase | 0.2 |
Prepare one or two extra tubes-worth of reagent (you'll use some liquid on the walls of your tips).
Once you've finished, you should run the resulting products on a gel to check if everything went as planned.
Tips
- Thaw the HF-Buffer, DMSO and dNTPs before making the mastermix.
- Avoid taking the Phusion-HF polymerase out of the freezer (only take it out briefly when you need to add it).
- If the reactions have different primers and/or template, add the polymerase right after the dNTPs, split the mastermix and add the rest.
- Don't forget positive and negative controls
- Primers should have similar Tms (less than 5°C).
- Primer Tm calculation is a less exact science than it should be (just test several tools and compare their results). If you're not sure what the correct Tm is, consider using a gradient PCR.
- Avoid primers with strong secondary structures.
- PCR can introduce mutations. Don't forget to sequence your final product (this could be your final plasmid): you really don't want to lose a few weeks because of a "corrupt" plasmid.
Protocol: Gel Electrophoresis
Agarose concentration depends on the size of the DNA to be run. We will mostly use 1%.
VOL is the desired volume of gel in ml:
CH Lab
- Add 0.01*VOL g of agarose to a clean glass bottle.
- Pour VOL/50 ml of 50xTAE in a graduated cylinder. Fill up to VOL ml with di water.
- Add the resulting VOL ml of 1xTAE to the glass bottle with agarose.
- Microwave, at 7, the bottle (loose cap!) until it boils.
- Carefully remove bottle (can be super heated!) and check for the total absence of particles. Microwave again if needed.
- Prepare a gel box, with comb, and fill it up with the agarose solution (maybe not the whole solution is needed).
- Add 0.05 µl per ml of gel in the box of Red Gel (it's in the iGEM drawer) and stirr until disolved.
- Wait until cold and solidified.
- Carefully remove comb.
- Place the box in the electrophoresis chamber.
- Fill up the electrophresis chamber with 1x TAE buffer.
- Add blue dye to the DNA samples (6x loading buffer, that is 10 µl in 50 µl of DNA solution).
- Inject 30 µl of ladder marker in the first well (that's 1 µg of DNA).
- Inject 60 µl of each DNA solution in the other wells.
- Set voltage to 70-90 V and run for 30-40 min, or until the dye reaches the last 25% of the gel length (DNA travels from - to +).
- Place the gel under the camera, cover, turn UV on and take photos!
Preparing the ladder:
- get 1kb ladder DNA from the freezer (500 µg/ml).
- for 30 charges, 30 µl per charge, we need 900 µl:
- 60 µl of 1kb ladder DNA
- 150 µl of dye (6x loading buffer)
- 690 µl of water
BM Lab
In this lab the gels are slightly different. The total volumes for the small, the medium and the large gel are respectively 60ml, 80ml and 90ml. As we use 0.5x TAE buffer instead of 1x, we can use higher voltages (170V seems to work fine). The gel should run 20-40 minutes, not more. As the gel is thinner, load less DNA (up to ~10ul).
Ran a PCR on the supposedly ligated colonies of TNFR-pGL and on every other plasmid of interest to add the restriction sites we want to them. Then ran a gel (CH lab version) of all the PCR products.
- Top of the gel
- Lane 1: 1kb ladder
- Lane 2: LovTAP1 (with primers that add the restriction enzyme sites to ligate it into the pMP plasmid)
- Lane 3: LovTAP2 (with primers that add the restriction enzyme sites to ligate it into the pMP plasmid)
- Lane 4: GFP (primers that add sites for pGL4.30)
- Lane 5: SEAP (primers that add sites for pGL4.30)
- Lane 6: SEAP (BioBricking primers)
- Lane 7: TNFR (primers that add sites for pGL4.30)
- Lane 8: TNFR (BioBricking primers)
- Lane 9: Master mix only (control)
- Lane 10: 1kb ladder
- Bottom of the gel
- Lane 1: 1kb ladder
- Lane 2: Supercoiled TNFR plasmid
- Lane 3: Ligation: TNFR2, colony b from 1:2 plate
- Lane 4: Ligation: TNFR2, colony c from 1:2 plate
- Lane 5: Ligation: TNFR2, colony a from 1:3 plate
- Lane 6: Ligation: TNFR1, colony b from 1:4 plate
- Lane 7: Ligation: TNFR1, colony c from 1:4 plate
- Gel image
- Comments
On the gel, we saw that most of our restriction site-adding PCRs had failed, and that the TNFR ligation results seemed weird at best, with several bands for each colony, all of them of quite different sizes. Later on, we found out this was due to a recurrent PCR machine malfunction.
