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Miniprep of pSB1C3-melanopsin
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 protocol (page 22) and a microcentrifuge.
The working PCR'd colonies were miniprepped and then sent for sequencing. The concentration was reasonable (around 200 ng/µl).
Colony PCR of pSB1C3-NFAT (Biobrick)
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 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.
- Comments
Gave no bands except for the positive control -> all the picked colonies are pSB1C3-RFP religations.
Maxiprep
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 here. We usually use the protocol that starts at page 24 for "Maxi".
Yesterday's maxiprep worked (500ul @ 400ng/ul). Not the best yield we've ever gotten, but it's be more than enough to start experimenting!
