Team:NTNU Trondheim/Protocols

From 2012.igem.org

(Difference between revisions)
 
Line 166: Line 166:
-
</div>
+
{{:Team:NTNU_Trondheim/Templates/Sponsors}}
-
</html>
+
-
{{:Team:NTNU_Trondheim/Templates/Footer}}{{:Team:NTNU_Trondheim/Templates/Sponsors}}
+
<html>
<html>
</div></div></html>
</div></div></html>
{{:Team:NTNU_Trondheim/Templates/Footer}}
{{:Team:NTNU_Trondheim/Templates/Footer}}

Latest revision as of 17:40, 25 September 2012

NTNU IS B.A.C.K.
Bacterial Anti-Cancer-Kamikaze

Protocols A list of recipes and laboratory procedures used by the team

Contents

Laboratory procedures


Transformation

Official iGEM transformation protocol: http://partsregistry.org/Help:Protocols/Transformation

We use this protocol with the following modifications:

The cells used are [http://products.invitrogen.com/ivgn/product/18265017 Subcloning Efficiency™ DH5α™ Competent Cells] from Life Technologies/Invitrogen

Inoculation after transformation

Using a sterile toothpick, pick/scratch a single colony from the transformants. Drop the toothpick into a plastic tube with 3 mL sterile liquid medium with the appropriate antibiotic(s). Close the tube but leave the cap slightly open to allow oxygen to enter, and incuate at 37 C with shaking.

DNA Isolation

We use the Promega Wizard Plus SV Minipreps DNA Purification System A1460 with the sentrifugation version of the [http://www.promega.com/~/media/files/resources/protocols/technical%20bulletins/0/wizard%20plus%20sv%20minipreps%20dna%20purification%20system%20protocol.pdf?la=en protocol] supplied by the vendor.

DNA Concentration measurements

Concentrations of DNA after isolation was measured with the [http://www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf NanoDrop ND-1000 Spectrophotometer].

Restriction digest

We are using the single reaction protocol from partsregistry.org : [http://partsregistry.org/Help:Protocols/Restriction_Digest]

Ligation

Ligation protocol from partsregistry.org [http://partsregistry.org/Help:Protocols/Ligation]:

Remember to do religation of backbone!!!!!!!!

Linearized plasmids

[http://partsregistry.org/Help:Protocols/Linearized_Plasmid_Backbones Official iGEM protocol]

Gel electrophoresis

Agarose with Gel Green is used when molding the gel. Choose an appropriate ladder as a referance to size of the fragments and put 2 µl of ladder in wells on either side of the samples. When applying the samples, add 20% loading dye to the sample. For instance, if the initial sample is 10 µl, add 2 µl loading dye. Apply the samples to individual wells in the gel.

Let the gel run for 45 min at 90 V, and if the bands are poorly separated, run a little longer. Beware that if the gel runs for a very long time, the smaller bands may move out of the gel.

Gel purification

We are using the QIAquick Gel Extraction kit, following the protocol from www.qiagen.com. [http://www.qiagen.com/literature/render.aspx?id=201083]

Preparation of samples for RNA isolation

RNA isolation

(RNAquenous kit from Ambion)

DNAse reaction

cDNA reaction


OD measurements

Unless otherwise noted, all OD measurements are made at 600 nm with a PerkinElmer Lambda 35 spectrometer, with un-inoculated medium as reference.

Fluorescence measurements

Fluorescence were measured with a Tecan Infinite M200 Pro microplate reader using Nunclon flat bottom black polystyrol 96 well plates.

Recipes


Growth media

LB-medium (LB-Lennox):

Antibiotics

Ampicillin stock solutions: 100 mg/mL dissolved in MQ water
Kanamycin stock solutions: 100 mg/mL dissolved in MQ water
Chloramphenicol: 34 mg/mL dissolved in 100% ethanol

Store at -20 C after preparation and between use.

Ampicillin media concentration: 100 ug/mL = 1 mL stock solution/L medium
Kanamycin media concentration: 100 ug/mL = 1 mL stock solution/L medium Chloramphenicol media concentration: 24 ug/mL = 0.7 mL stock solution/L medium

When transforming low copy number plasmids, it may be necessary to reduce the antibiotic to allow for the reduced antibiotic resistance of the tranformant.

Glycerol stocks

Prepare a solution of 60 % glycerol in water. Add 400 uL glycerol solution and 800 uL of the culture to be stored in a cryogenic tube. Place in 5 C refrigerator for 30 min, then move to -80 C freezer.

Cake

Batter is prepared following the protocol by Kakemonsen ([http://www.kakemonsen.no/sider/vis.asp?id=1186 1]). Sliced pieces of Malus domestica is inserted into the batter, while sucrose and ground Cinnamomum verum bark is mixed 2:1 by volume and distributed evenly on top of the batter, which is placed in a 26 cm diameter open-top vessel. The mixture is incubated in a dry air oven at ~180 C for 45 min.



Retrieved from "http://2012.igem.org/Team:NTNU_Trondheim/Protocols"