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Team:BIOSINT Mexico/Protocols
From 2013.igem.org
(Difference between revisions)
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| + | '''Protocol 1: Preparation of growth mediums (MRS and LB)''' | ||
| + | |||
| + | *Weight 18.6 g of Agar MRS to prepare 300ml of medium | ||
| + | *Weight 7 g of Agar LB to prepare 200ml of medium | ||
| + | *Dilute 5 g of A-MRS in 80 ml of distilled water | ||
| + | *Add 5 g and 170 ml of distilled water | ||
| + | *Dilute 7 g of Agar LB in 100ml of distilled water | ||
| + | *Add 100ml of distilled water | ||
| + | *Place the autoclave tape | ||
| + | *Set the autoclave | ||
| + | *When the temperature of the autoclave reaches 120⁰c, cut the power by half | ||
| + | *When the temperature reaches 105⁰c, open the valve. | ||
| + | *Retrieve the mediums | ||
| + | |||
| + | |||
| + | ---- | ||
| + | '''Protocol 2: Preparation of MRS broth and glycerol stocks''' | ||
| + | |||
| + | *Weight 0.7679 g of powder for MRS broth | ||
| + | *Measure 15ml of distilled water in a 100ml measuring cylinder | ||
| + | *Weight 10.009g of powder for MRS broth | ||
| + | *Dilute it in 100ml of distilled water | ||
| + | *Add 100ml of distilled water | ||
| + | *Dilute 0.7679g of powder for MRS broth in 15ml of distilled water | ||
| + | *Mark the flasks with autoclave tape and sterilize them for 15 minutes at 121⁰c | ||
| + | *Label the broth | ||
| + | *Mix the 15ml of MRS broth and the 15ml of glycerol to 100% in the laminar flow cabinet. Shake them gently until a homogenous mixture is obtained in a falcon tube of 45ml | ||
| + | *Add an aliquot of L. Plantarum to the broth and glycerol solution and seal it with parafilm | ||
| + | *Incubate it at 37⁰c and 200rpm | ||
| + | *After 24 hours, take the sample out of the incubator to striate it | ||
| + | *Place 2500 (x2)ml in 22 eppendorf tubes and using a micropipette of 200µl , cultivate and recompile the mixture of L. Plantarum contained in the falcon tube | ||
| + | *Centrifuge 1 eppendorf tube at 13.4 rpm for 3 minutes | ||
| + | *Discard the supernatant and add a solution of MRS broth (500ml) | ||
| + | *Once the mixture is made, striate two petri dishes with MRS Agar in the laminar flow cabinet | ||
| + | *Place the petri dishes facing down in the incubator at 37⁰c and 200 rpm | ||
| + | *Store the remaining samples (21 falcon tubes) in deep freezing | ||
| + | *Striate E. Coli K12 in two petri dishes with LB Agar | ||
| + | *Place the dishes in the incubator at 37⁰c. | ||
| + | |||
| + | |||
| + | ---- | ||
| + | '''Protocol 3: Plasmid purification''' | ||
| + | |||
| + | *Centrifuge an eppendorf tube of 1.5ml with L. Plantarum culture at 12000 rpm for 1 minute, two times | ||
| + | *Discard the supernatant and resuspend the tube’s content with more L. Plantarum culture in MRS broth | ||
| + | *Centrifuge the eppendorf tube for 15min at 6000 rpm | ||
| + | *Discard the supernatant | ||
| + | *Resuspend the pellet in 250ml resuspension buffer | ||
| + | *Add 250ml of Lysis Buffer L7 | ||
| + | *Gently mix the tube carefully inverting it 5 times carefully | ||
| + | *Add and mix softly 350ml of precipitation Buffer N4, inverting the tube | ||
| + | *Centrifuge it at 12000 rpm for 10 minutes | ||
| + | *Transfer the supernatant into a spin column inside a washtube | ||
| + | *Centrifuge it at 12000 rpm for a minute | ||
| + | *Discard the supernatant and add 500 ml of Wash Buffer with ethanol (w10) to the column | ||
| + | *Incubate it for one minute at room temperature | ||
| + | *Centrifuge the column at 12000 rpm for 1 minute | ||
| + | *Discard the liquid from the washtube and place the column inside the tube | ||
| + | *Add 700ml of Wash Buffer W9 with ethanol to the column | ||
| + | *Centrifuge the column with the washtube at 12000 rpm for 1 minute | ||
| + | *Discard the liquid from the washtube | ||
| + | *Centrifuge the column with the washtube at 12000 rpm for 1 minute | ||
| + | *Discard the liquid from the washtube | ||
| + | *Place the column inside an eppendorf tube of 1.5ml | ||
| + | *Add 75ml of preheated TE Buffer at the center of the column | ||
| + | *(Warm the TE Buffer previously in water bath at 65⁰c-70⁰c for 3 minutes) | ||
| + | *Incubate the column for 1 minute at room temperature | ||
| + | *The column was centrifuged at 12000 rpm for 2 minutes | ||
| + | *(The eppendorf tube contains the purified plasmid) | ||
Revision as of 01:02, 28 September 2013
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Protocols
Protocol 1: Preparation of growth mediums (MRS and LB)
- Weight 18.6 g of Agar MRS to prepare 300ml of medium
- Weight 7 g of Agar LB to prepare 200ml of medium
- Dilute 5 g of A-MRS in 80 ml of distilled water
- Add 5 g and 170 ml of distilled water
- Dilute 7 g of Agar LB in 100ml of distilled water
- Add 100ml of distilled water
- Place the autoclave tape
- Set the autoclave
- When the temperature of the autoclave reaches 120⁰c, cut the power by half
- When the temperature reaches 105⁰c, open the valve.
- Retrieve the mediums
Protocol 2: Preparation of MRS broth and glycerol stocks
- Weight 0.7679 g of powder for MRS broth
- Measure 15ml of distilled water in a 100ml measuring cylinder
- Weight 10.009g of powder for MRS broth
- Dilute it in 100ml of distilled water
- Add 100ml of distilled water
- Dilute 0.7679g of powder for MRS broth in 15ml of distilled water
- Mark the flasks with autoclave tape and sterilize them for 15 minutes at 121⁰c
- Label the broth
- Mix the 15ml of MRS broth and the 15ml of glycerol to 100% in the laminar flow cabinet. Shake them gently until a homogenous mixture is obtained in a falcon tube of 45ml
- Add an aliquot of L. Plantarum to the broth and glycerol solution and seal it with parafilm
- Incubate it at 37⁰c and 200rpm
- After 24 hours, take the sample out of the incubator to striate it
- Place 2500 (x2)ml in 22 eppendorf tubes and using a micropipette of 200µl , cultivate and recompile the mixture of L. Plantarum contained in the falcon tube
- Centrifuge 1 eppendorf tube at 13.4 rpm for 3 minutes
- Discard the supernatant and add a solution of MRS broth (500ml)
- Once the mixture is made, striate two petri dishes with MRS Agar in the laminar flow cabinet
- Place the petri dishes facing down in the incubator at 37⁰c and 200 rpm
- Store the remaining samples (21 falcon tubes) in deep freezing
- Striate E. Coli K12 in two petri dishes with LB Agar
- Place the dishes in the incubator at 37⁰c.
Protocol 3: Plasmid purification
- Centrifuge an eppendorf tube of 1.5ml with L. Plantarum culture at 12000 rpm for 1 minute, two times
- Discard the supernatant and resuspend the tube’s content with more L. Plantarum culture in MRS broth
- Centrifuge the eppendorf tube for 15min at 6000 rpm
- Discard the supernatant
- Resuspend the pellet in 250ml resuspension buffer
- Add 250ml of Lysis Buffer L7
- Gently mix the tube carefully inverting it 5 times carefully
- Add and mix softly 350ml of precipitation Buffer N4, inverting the tube
- Centrifuge it at 12000 rpm for 10 minutes
- Transfer the supernatant into a spin column inside a washtube
- Centrifuge it at 12000 rpm for a minute
- Discard the supernatant and add 500 ml of Wash Buffer with ethanol (w10) to the column
- Incubate it for one minute at room temperature
- Centrifuge the column at 12000 rpm for 1 minute
- Discard the liquid from the washtube and place the column inside the tube
- Add 700ml of Wash Buffer W9 with ethanol to the column
- Centrifuge the column with the washtube at 12000 rpm for 1 minute
- Discard the liquid from the washtube
- Centrifuge the column with the washtube at 12000 rpm for 1 minute
- Discard the liquid from the washtube
- Place the column inside an eppendorf tube of 1.5ml
- Add 75ml of preheated TE Buffer at the center of the column
- (Warm the TE Buffer previously in water bath at 65⁰c-70⁰c for 3 minutes)
- Incubate the column for 1 minute at room temperature
- The column was centrifuged at 12000 rpm for 2 minutes
- (The eppendorf tube contains the purified plasmid)
