Team:NTNU Trondheim/Protocols

From 2012.igem.org

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__TOC__
__TOC__
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==Transformation==
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==Lab procedures==
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===Transformation===
Official iGEM transformation protocol: http://partsregistry.org/Help:Protocols/Transformation
Official iGEM transformation protocol: http://partsregistry.org/Help:Protocols/Transformation
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The cells used are [http://products.invitrogen.com/ivgn/product/18265017 Subcloning Efficiency™ DH5α™ Competent Cells] from Life Technologies/Invitrogen
The cells used are [http://products.invitrogen.com/ivgn/product/18265017 Subcloning Efficiency™ DH5α™ Competent Cells] from Life Technologies/Invitrogen
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==Inoculation after transformation==
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===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.
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.
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==DNA Isolation==
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===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.
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.
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==DNA Concentration measurements==
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===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].
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].
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==Restriction digest==
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===Restriction digest===
We are using the single reaction protocol from partsregistry.org : [http://partsregistry.org/Help:Protocols/Restriction_Digest]
We are using the single reaction protocol from partsregistry.org : [http://partsregistry.org/Help:Protocols/Restriction_Digest]
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*Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.
*Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.
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==Ligation==
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===Ligation===
Ligation protocol from partsregistry.org [http://partsregistry.org/Help:Protocols/Ligation]:
Ligation protocol from partsregistry.org [http://partsregistry.org/Help:Protocols/Ligation]:
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Remember to do religation of backbone!!!!!!!!
Remember to do religation of backbone!!!!!!!!
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==Linearized plasmids==
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===Linearized plasmids===
[http://partsregistry.org/Help:Protocols/Linearized_Plasmid_Backbones Official iGEM protocol]
[http://partsregistry.org/Help:Protocols/Linearized_Plasmid_Backbones Official iGEM protocol]
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==Gel electrophoresis==
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===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.
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.
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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.
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.
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==Gel purification==
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===Gel purification===
We are using the QIAquick Gel Extraction kit, following the protocol from www.qiagen.com.
We are using the QIAquick Gel Extraction kit, following the protocol from www.qiagen.com.
[http://www.qiagen.com/literature/render.aspx?id=201083]
[http://www.qiagen.com/literature/render.aspx?id=201083]
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==Preparation of samples for RNA isolation==
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===Preparation of samples for RNA isolation===
* Grow overnight cultures
* Grow overnight cultures
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* Spin down at 6000 g for 10 minutes in 4 &deg;C
* Spin down at 6000 g for 10 minutes in 4 &deg;C
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==RNA isolation==
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===RNA isolation===
(RNAquenous kit from Ambion)
(RNAquenous kit from Ambion)
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* Measure concentrations on NanoDrop
* Measure concentrations on NanoDrop
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==DNAse reaction==
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===DNAse reaction===
* Calculate the volume of RNA solution necessary to obtain a total of 3000 ng RNA
* Calculate the volume of RNA solution necessary to obtain a total of 3000 ng RNA
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* Incubate for 10  minutes at 65 &deg;C
* Incubate for 10  minutes at 65 &deg;C
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==cDNA reaction==
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===cDNA reaction===
* Prepare bulk mixture:
* Prepare bulk mixture:
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* If qPCR is not to be performed immediately, the cDNA samples should be frozen down at -80 &deg;C
* If qPCR is not to be performed immediately, the cDNA samples should be frozen down at -80 &deg;C
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==Mastermix for real time PCR==
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<!--===Master mix for real time PCR===
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Coming soon...
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Coming soon...-->
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==OD measurements==
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===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.
Unless otherwise noted, all OD measurements are made at 600 nm with  a PerkinElmer Lambda 35 spectrometer, with un-inoculated medium as reference.
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==Fluorescence measurements==
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===Fluorescence measurements===
Fluorescence were measured with a Tecan Infinite M200 Pro microplate reader using Nunclon flat bottom black polystyrol 96 well plates.
Fluorescence were measured with a Tecan Infinite M200 Pro microplate reader using Nunclon flat bottom black polystyrol 96 well plates.

Revision as of 14:21, 24 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


Lab procedures

Transformation

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

We use this protocol with the following modifications:

The cells used are 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 protocol supplied by the vendor.

DNA Concentration measurements

Concentrations of DNA after isolation was measured with the NanoDrop ND-1000 Spectrophotometer.

Restriction digest

We are using the single reaction protocol from partsregistry.org : [1]

Ligation

Ligation protocol from partsregistry.org [2]:

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

Linearized plasmids

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. [3]

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 (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. </div> </html>

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