"
Contents |
Transformations
Protocol: E.Coli Transformation
- Thaw the competent E.coli (DH5alpha) cells on ice (not in hands!)
- As soon as it is thawed, add 50µl of the cells to the DNA (~50-100 ng of pure plasmid, or some 2 µl usually)
- Let it rest on ice for 20-30 min. Meanwhile, put agar plate (with correct antibiotic) at 37°C for prewarming.
- Put the tube with DNA+E.coli at 42°C for 45 sec - 1 min (heat shock)
- Add 400 µl of LB broth and place at 37°C for 20-30 min (shaking)
- Spread the cells on the prewarmed plate (and let it dry)
- Incubate the plate upside-down at 37°C for ~14-15 hours (leaving it more than 16h decreases the plasmid quality)
Early in the morning, bacteria were transformed with the pGL-SEAP ligation result from the day before, but also with the pCEP4 and the pcDNA3.1(-) plasmids (future backbones for the readout for LovTAP, to create a stock of the plasmids). They were incubated for as long as we could allow.
Melanopsin and LovTAP readout Biobrick PCR
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.
The biobricking primers for melanopsin gene and LovTAP readout construct arrived and we could perform a PCR to get the inserts for ligation into the Biobrick standard plasmid pSB1C3.
Templates:
- pHY42 (melanopsin)
- RO (Nicolas Gobet's plamid)
Results
- Comments
All the reactions worked for melanopsin. Therefore we used the PCR products right away for digestion and ligation. For the LovTAP readout, none of the reactions worked, so it has to be redone with another temperature gradient.
LovTAP Maxiprep (!) overnight culture
Protocol: MaxiPrep
The evening before, take a big Erlenmeyer (at least 1L) and put 200ml LB in it. Add the appropriate antibiotics at the correct concentration (ampicilin: 200ul of 100mg/ml solution). Put in bacteria from a single colony of a freshly streaked plate or from a glycerol stock (warning: taking bacteria from glycerol stock seems to cause them to start growing later - due to thawing? - add one-two hours to the incubation time). Put them in the incubator for 14-15 hours (the contents of the bottle should be yellow-ish between translucid and opaque).
We then use the MaxiPrep kit (Plasmid DNA Purification kit) and protocol from Macherey-Nagel.
The complete handbook can be found [http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NuBo.pdf here]. We usually use the protocol that starts at page 24 for "Maxi".
A pcDNA3.1(+)-LovTAP colony was grown in an erlenmeyer overnight in the shaker, for future mammalian cell transfections.
Overnight miniprep cultures of the transformed bacteria
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.
In the evening, we saw colonies of bacteria transformed in the morning. We made miniprep cultures out of them.
Ligation of LovTAP-BB with pcDNA3.1(-)
Protocol: Ligation
Ligation is a method of combining several DNA fragments into a single plasmid. This is often the
step following a PCR (and a PCR cleanup) or a gel extraction. You can also do a "dirty" ligation, where you follow a certain number of digestions directly by a ligation.
- Download the following spreadsheet : File:Team-EPF-Lausanne Ligation.xls
- Fill in the pink areas with the vector and fragment concentration, their size and the ratio.
- Add all the suggested ingredients order in a microcentrifuge tube, in the order they appear.
- Ligate for 2 hours at 14ºC.
- Immediately transform competent bacteria with the ligation product.
Note: This protocol hasn't been optimized for blunt-end ligation (though it might still work).
The previously digested vector and BioBrick template were ligated together and plated.
