"
Contents |
Colony PCR of pcDNA3.1(+)-LovTAP (Part 1)
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.
Plan : Use T7 + BGH and T7 + LovTAP check primer sets to check the presence of LovTAP in our colonies and verify that the backbone is pcDNA3.1(+) and not LovTAP's former backbone pMA (determined by the presence or not of a polyA signal BGH, which is absent in pMA).
Since the Tm for T7 was nowhere to be found, a gradient PCR was made to figure it out. [insert picture of gel here and maybe protocol]
Colony preparation: The four plates were labeled
- A (pcDNA3.1-LovTAP unpurified 1:2)
- B (pcDNA3.1-LovTAP unpurified 1:3)
- C (pcDNA3.1-LovTAP purified 1:3)
- Control (pcDNA3.1 only)
Of each one we decided to pick 8 colonies, except for the control where we picked only one.
- 5-10ul of Lyse and Go was added into all the destination PCR tubes
- 3ml of LB + 80ul of 100mg/ml Amp in the culture tubes
- Colonies were picked, dipped into the culture tube and then into the PCR tube.
- Culture tubes were incubated at 37°C and PCR tubes stored in Matt's fridge until next morning.
Melanopsin readouts PCR product digestion
Protocol: Quick and Dirty Digestion
Since Nanodrop concentration results for PCR and samples extracted from gels have proved unreliable, we have arrived at a sort of a default protocol for DNA of an uncertain concentration.
For a ligation or when large quantities of DNA are needed:
- DNA 15 microliters
- Restriction enzyme 1 @ 100x 1 microliters
- Restriction enzyme 2 @ 100x 1 microliters
- BSA (if needed) @ 10 x 5 microliters
- Demineralized water 28 microliters
For checking the presence of a restriction site on a gel:
- DNA 5 microliters
- Restriction enzyme 1 @ 100x 1 microliters
- Restriction enzyme 2 @ 100x 1 microliters
- BSA (if needed) @ 10x 5 microliters
- Demineralized water 38 microliters
The total volume should be 50 microliters
Incubation should ideally be at 37°C. When in doubt on the quantity of DNA used, favor a longer (20 to 30 minutes longer) incubation time than what is indicated on the NEB double digest guide. Avoid enzyme pairs with different incubation temperatures. This doubles the incubation time.
After the incubation period, the enzymes need to be heat-inactivated. Heat inactivation temperatures are usually on the order of 80 C. 20 minutes usually does the job.
The samples can then be frozen if needed.
The following PCRs worked at Matt's lab: SEAP, TNFR and eGFP (also used as a control at a Tm of 55°C). Therefore these products were ready to be digested in order to be ligated into pGL4.30 to achieve the cloning of the three Fussenegger experiment readouts. Digestion:
- TNFR and eGFP with HindIII and FseI
- SEAP with HindIII and MfeI
Cleanup of the PCR products
Protocol: PCR Cleanup
After doing a PCR, the resulting DNA should be cleaned up to get
rid of the primers, polymerases, dNTPs and the various other reagents
used in the PCR. This can also be used to remove small fragments
of DNA from other sources (such as digestions).
We used Macherey-Nagel's "Nucleospin® Gel and PCR clean-up" kit. The manual can be found here: [http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf Gel and PCR clean-up Manual]
The kit uses a silica membrane to bind DNA which is then washed with several different buffers. The final step is the removal from the membrane by elution and recovery of our cleaned DNA.
Note: To increase the yield we applied the optional steps in 4 and 5 in the PCR cleanup protocol on pages 18 and 19. By incubating the columns at 70 degrees and eluting the DNA with heated elution buffer the yield of longer fragments can be increased.
Two separate tubes were digested for each readout, one of which was purified (PCR clean up kit protocol) afterwards. That way we will be able to compare the results between ligation with or without previous PCR clean up and see if it's a necessary step or not.
Nanodrop of the cleaned PCR products
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 us low concentrations, as usual (10-20 ng/µl). Maybe the ligations are going to be more successful if the DNA is pure.
LovTAP readout digestion
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.
In order to clone the LovTAP readout into a mammalian expression vector (either pcDNA3.1(-) or pCEP4) we can simply cut it out with KpnI and BamHI. Alternatively, KpnI and XhoI also work but only for pcDNA3.1(-) insertion. So two different digestions were made on the Maxiprep of the LovTAP readout (RO):
- KpnI + BamHI
- KpnI + XhoI
Gel electrophoresis
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).
The two products were run on a 1% agarose gel.
- Gel image
Gel extraction
Protocol: Gel Extraction
A gel extraction is used to select a fragment of DNA of a specific length
out of a solution composed of different fragments (ideally the difference
in length between the wanted fragment and the closest-sized fragment should
be more than 200bp). These fragments are often obtained after a
digestion.
The yeild for this procedure is typically very poor so a large amount of starting material, digested DNA in this case is required. We typically used 4 micrograms. The digestion products are loaded on a gel. Lanes on both sides of the one to be extracted should be empty to make cutting easier and avoid contamination with other fragments.
The gel should be run long enough for the bands to be spread out. This is particularly important if the fragment of interest is around the same length as other expected digestion products. UV light is necesary to observe the bands on the gel but exposure time should be minimized to avoid DNA damage. The fragment of interest is then excise and put in an Eppendorf (consider using a 2ml one).
To extract the DNA from the agarose we used Macherey-Nagel's "Nucleospin® Gel and PCR clean-up" kit. Its manual can be found here: [http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf Gel and PCR clean-up Manual]
Tips
- Cut away as much Agar as possible without slicing into the DNA. Excess agar will require more solvent to dissolve and will result in a poorer yeild upon elution.
- Minimize the DNA's exposure to the UV-light. UV will damage DNA and have negative effects on any subsequent reactions (for example, ligations can be 10'000x less effective when DNA has been exposed to too much UV light [http://openwetware.org/wiki/DNA_Ligation]
The lower band was then cut out and purified with the gel extraction kit following the protocol in the manual.
Agar plate preparation
Protocol: Agar Plates
- Add to a bottle:
- 20 g/l of LB broth powder.
- 10 g/l of Agar (not agarose).
- Fill up with DI water and autoclave (program 106, takes 2 hours). Remember to leave the cap loose!
- Label the plates (found in the stock room).
- When the autoclaving is done, close the bottle cap and take the bottle out.
- Let rest until it cools down to around 55ºC (can be held for some seconds): if warmer, the antibiotic degrades, if colder, the broth gelifies.
- Add the antibiotic (Ampicillin 100 µg/ml) = 1 ml of 100 mg/ml Amp for 1l of broth. Same for chloramphenicol or Spe.
- Add 25-30 ml of broth to each plate and let open for 2 hours.
- Close plates, wrap in alu foil (Amp is sensitive to light) and store at +4ºC.
We used all the Ampicillin agar plates for previous transformation, therefore we had to prepare new ones.
We prepared 33 plates.
