Team:EPF-Lausanne/Notebook/12 September 2012

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== Western blot, day 1 ==
 
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== Melanopsin Biobrick colony PCR ==
== Melanopsin Biobrick colony PCR ==

Revision as of 15:34, 26 September 2012



Contents

Melanopsin Biobrick colony 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.


Colony PCR was performed on the lysed colonies from the day before, 14 from the pSB1C3-Melanopsin plate and 2 from the control plate.

Positive control: PHY42+: PHY42 with T7_fwd & melanopsin_bb_rev Expected size : Around 1600bp

Negative controls: pBS1C3 - = pSB1C3 with pSB1C3_check_fwd & melanopsin_bb_rev PHY42- = PHY42 (melanopsin) with pSB1C3_check_fwd & melanopsin_bb_rev

PCR with HF-Buffer (5x) and DMSO at 63°C with 1ul colony lysate as template.

Results:

Team-EPF-Lausanne-melanopsin-bb-pcr.png

LovTAP Readout Maxiprep

Team:EPF-Lausanne/Protocol/Maxiprep

Comments

Insert comments about what happened.

NFAT gel extraction

Protocol: None

Forgot to insert protocol.


PCR products from three NFAT pcr reaction were digested w/ ecoRI and speI for ligation and then the cleanup was done on a gel due to competeing pcr reaction being present. This allowed for a cleaner product for our ligation fragment.


Team epfl NFAT gel extraction.jpg

Maxiprep

Yesterday's maxiprep failed... again! (the pellet disappeared during the proces... must've aspirated it somehow).

Started yet another maxiprep of the Read-out plasmid.


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".