Team:NTNU Trondheim/Protocols

From 2012.igem.org

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{{:Team:NTNU_Trondheim/Templates/Header}}
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<html>
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<span class="heading">Protocols<hr/></span>
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<div class="page-header-top">
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This is a list of recipes and protocols used by the team.
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<h1>Protocols <small> A list of recipes and laboratory procedures used by the team</small></h1>
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__TOC__
__TOC__
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==Transformation==
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==Laboratory procedures==
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----
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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]
*250 ng of DNA is added together with the appropriate amount of dH2O, for a total volume of 16 uL.
*250 ng of DNA is added together with the appropriate amount of dH2O, for a total volume of 16 uL.
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*Add 2,5 uL of NED-buffer 2
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*Add 2,5 uL of the appropriate NEBuffer
*Add 0,5 uL og BSA
*Add 0,5 uL og BSA
*Add 0,5 uL of Enzyme 1
*Add 0,5 uL of Enzyme 1
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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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*Incubate for 30min at 16C and 20min at 80C to heat kill
*Incubate for 30min at 16C and 20min at 80C to heat kill
*Use 2ul of ligation to transform into competent cells
*Use 2ul of ligation to transform into competent cells
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<!--$frac{kb insert\cdotC_{vector}\cdot\mu L vector\cdot ratio}{kb vector\cdot C_{insert}}=\mu L insert$-->
<!--$frac{kb insert\cdotC_{vector}\cdot\mu L vector\cdot ratio}{kb vector\cdot C_{insert}}=\mu L insert$-->
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==Gel electrophoresis==
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Remember to do religation of backbone!!!!!!!!
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===Linearized plasmids===
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[http://partsregistry.org/Help:Protocols/Linearized_Plasmid_Backbones Official iGEM protocol]
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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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==OD measurements==
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===Preparation of samples for RNA isolation===
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* Grow overnight cultures
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* Mix 1 ml cell culture with 2 ml RNA-protect (QIAGEN)
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* Vortex for 5 seconds
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* Incubate for 5 minutes in room temperature
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* Spin down at 6000 g for 10 minutes in 4 &deg;C
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===RNA isolation===
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(RNAquenous kit from Ambion)
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* Add 100 µl Lysozyme/TE-mix to each sample
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* Incubate for 5 minutes in room temperature
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* Add 300 µl lysis/binding solution
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* Vortex to make sure everything is solved
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* Add 400 µl water for 64 % ethanol
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* Turn the tubes 4 times, and transfer to filtertubes
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* Centrifuge for 1 minute at 13000 g
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* Add 700 µl wash solution 1
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* Centrifuge for 1 minute at 13000 g
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* Add 500 µl wash solution 2/3
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* Centrifuge for 1 minute at 13000 g
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* Add 500 µl wash solution 2/3
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* Centrifuge for 1 minute at 13000 g
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* Centrifuge for an additional minute at 13000 g
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* Add 40 µl preheated elution buffer
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* Centrifuge for 1 minute at 13000 g
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* Add 20 µl preheated elution buffer
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* Centrifuge for 1 minute at 13000 g
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* Measure concentrations on NanoDrop
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===DNAse reaction===
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* Calculate the volume of RNA solution necessary to obtain a total of 3000 ng RNA
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* Add SIV to 25 µl
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* Add 2.7 µl DNAse buffer and 1 µl DNAseI
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* Incubate for 30 minutes at 37 &deg;C
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* Add 5 µl inactivation mixture and flip the tubes
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* Incubate for 2 minutes in room temperature
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* Centrifuge for 2 minutes at 13000 g
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* Transfer 2 µl of the supernatant to a new tube and add 18 µl SIV
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* Incubate for 10  minutes at 65 &deg;C
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===cDNA reaction===
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* Prepare bulk mixture:
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** 5 µl bulk reaction mixture
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** 1 µl RNA primers
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** 1 µl DTT
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* Mix 4 µl sample with 3.5 µl bulk mixture
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* Incubate for 1 hour at 37 &deg;C
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* If qPCR is not to be performed immediately, the cDNA samples should be frozen down at -80 &deg;C
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<!--===Master mix for real time PCR===
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Coming soon...-->
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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.
==Recipes==
==Recipes==
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===Growth media===
===Growth media===
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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.
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.
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===Cake===
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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.
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{{: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.



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