Team:Copenhagen/Protocol

From 2011.igem.org

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(→Ligation Protocol)
(→Ligation Protocol)
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==Ligation Protocol==
==Ligation Protocol==
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How to ligate a BioBrick into a plasmid backbone. Use 3 times insert (BioBrick) to 1 time plasmid (3:1) in molar amounts. In calculating the amounts of DNA consider the length of the BioBrick and the plasmid. Use 25-30 ng plasmid.
+
How to ligate a BioBrick into a plasmid backbone. Use 3 times insert (BioBrick) to 1 time plasmid (3:1) in molar amounts. In calculating the amounts of DNA consider the length of the BioBrick and the plasmid. Use 25-30 ng plasmid. Note: iGEM are ligating eqimolar amounts of insert and plsmid as the only one in the world.
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Following the two different approaches of digestion, where you obtain different concentrations of the DNA, use either iGEM or Kenneths procedure for ligation. See below.  
'''Example'''
'''Example'''
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2 uL digested plasmid (12,5 ng/uL)
2 uL digested plasmid (12,5 ng/uL)
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3,7 uL digested BioBrick (10 ng/uL)
+
x uL digested BioBrick (10 ng/uL)
 +
 
 +
1 uL T4 Ligase buffer
 +
 
 +
0,5 uL T4 DNA Ligase
 +
 
 +
Add water uptil 10 uL
 +
 
 +
 
 +
'''Kenneth'''
If you have used Kenneths digestion approach, you have to determine the concentrations of the BioBrick and plasmid by NanoDrop.
If you have used Kenneths digestion approach, you have to determine the concentrations of the BioBrick and plasmid by NanoDrop.
 +
 +
 +
x uL gel extracted plasmid (25-30 ng)
 +
 +
x uL gel extracted BioBrick
 +
 +
2 uL T4 ligase buffer
 +
 +
0,5 uL T4 DNA ligase
 +
 +
Add water uptil 20 uL
 +
 +
 +
Ligate at room temperature in 10 min.
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Keep on ice until transformation.
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==Transformation protocol for pSB1C3==
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The only exeption to the procedure is an incubation step after adding the LB medium. Incubation at 1 hr. at 37 degrees while shaking.

Revision as of 16:43, 13 July 2011


Contents

Protocols

First Week 22-24 of June

Mutations

Ingredients

Site directed mutagenesis of 3 plant CYP450 (79A1, 79A2, 79B1).

We aim to destroy restrictionenzymes recognitionsites.

Primers was supplied by IDT


10 μl of 5× reaction buffer

X μl (50 ng) of dsDNA template

X μl (125 ng) of oligonucleotide primer #1

X μl (125 ng) of oligonucleotide primer #2

1 μl of dNTP mix

ddH2O to a final volume of 50 μl

Then add

1 μl of X7 fusion DNA polymerase


Poly Chain Reaction

We ran a PCR to syntesise and amplify our mutated CYP's.


Cycling Parameters for the QuikChange Site-Directed Mutagenesis Method

Cycles 12

Temperature 98°C Time 30 seconds

Temperature 55°C Time 1 minute

Temperature 72°C Time 30 seconds/kb of plasmid length


Digestion

We aim to remove the parentel CYP.

We take advantage of the fact that this CYP is methylated on cytosines. Dpn is a restriction enzyme that cuts DNA which is methylated - therefore our new mutated CYPs remain untouched.

1. Add 1 μl of the Dpn I restriction enzyme (10 U/μl) directly to each amplification reaction.

2. Gently and thoroughly mix each reaction mixture by pipetting the solution up and down several times. Spin down the reaction mixtures in a microcentrifuge for 1 minute and immediately incubate each reaction at 37°C (in a heater with lid or in a 37°C room) for 1 hour to digest the parental (i.e., the nonmutated) supercoiled dsDNA.


Source

Adapted from QuikChange™ Site-Directed Mutagenesis Kit INSTRUCTION MANUAL

Competent Cells

E. coli Calcium Chloride competent cell protocol

1. Inoculate a single colony into 5mL Lb in 15mL falcon tube. Grow O/N at 37°C.

2. Use 1mL to inoculate 100mL of LB in 250mL bottle the next morning.

3. Shake at 37°C for 1.5-3hrs until OD600 = 0.4-0.8

Then…. 1. Put the cells on ice for 10 mins (keep cold form now on).

2. Collect the cells by centrifugation in the big centrifugue for 10 mins at 6krpm

3. Decant supernatant and gently resuspend on 10 mL cold 0.1M CaCl (cells are susceptible to mechanical disruption, so treat them nicely).

4. Incubate on ice x 20 mins

5. Centrifuge as in 2

6. Discard supernatant and gently resuspend on 5mL cold 0.1MCaCl/15%Glycerol (from a 85% stock)

7. Dispense in microtubes (300μL/tube). Freeze in -80°C.

Source:

Adapted from http://www.med.nyu.edu/medicine/labs/blaserlab/Protocols/E-coli_competent_cells_protocol_&_transformation.pdf


LBamp Plates

1. 500 ml LB agar and 500 μL amphicilin 2. Pour on plates 3. Leave with the lid half on for 30 minutes at room temperature 4. Put in refrigerator until needed.

Transformations

Transformation of Ca++ competent cells

1. Put ~50μL of competent cells to prechilled microtubes. Wait 1 minute. Add 10μL of circular plasmid or all of a ligation reaction of plasmid DNA.

2. Incubate for 15 mins on ice.

3. Heat shock for 30 seconds at 42°C. Put back on ice.

4. Add 70 μL LB

4. Plate the whole lot in LBamp plates

5. Leave the plates at 37°C O/N

Second Week

Mini prep

Ingredients

QIAprep® Spin Miniprep Kit

Miniprep

1. Pellet 5 ml bacterial overnight culture by centrifugation at 4500 rpm for 10 min at room temperature (20°C).

2. Resuspend pelleted bacterial cells in 500 μl Buffer P1 and transfer and divide in two microcentrifuge tubes.

3. Add 250 μl Buffer P2 to each tube and mix thoroughly by inverting the tube 4–6 times until the solution becomes blue. Do not allow the lysis reaction to proceed for more than 5 min.

4. Add to each 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. With LyseBlue reagent, the solution will turn colorless.

5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.

6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 60 s and discard the flow-through.

7. Wash the QIAprep spin column by adding 0.5 ml Buffer PB. Centrifuge for 60 s and discard the flow-through

8. Wash the QIAprep spin column by adding 0.75 ml Buffer PE. Centrifuge for 60 s and discard the flow-through, Transfer the QIAprep spin column to the collection tube.

9. Centrifuge for 1 min to remove residual wash buffer.

10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of the QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

Adapted from

Quick-StartProtocol QIAprep® Spin Miniprep Kit October 2010 [http://www.qiagen.com/literature/render.aspx?id=201081]

Restrictionsite analysis

To confirm that our mutations worked we analyses the purified plasmids by cutting them with the restrictionenzyme whose recognitionsite we aim to remove.

Ingrediens

1μl BSA (10x) diluted ca. 500ng Plasmid 1μl Restrictionenzyme 1μl NEBuffer Water up to 10μl

Cut at 37 degress for 1 hour.

Add 2,5μl loading buffer to the digestion

Run it on a gel

Visualize it and hope to see just one band. If two bands are present the mutation has not worked. (The original plasmid conatined a restrictionsite in the vector as well as in the CYP)

Third Week

PCR Reaction

We add prefix and suffix to our newly made BioBricks with PCR


Ingredients


Water to a final volume of 50μl

10 μl 5x Pfusion HF buffer

1 μl 10mM dNTP

0,5μM Primer A

0,5μM Primer A

0,5 μl template (ca. 150 ng Template)

0,50 μl Pfusion X7 Polymerase


PCR


Initial denaturation 98°C


Cycles 25

Denaturation Temperature 98°C Time 10 seconds

Anneal Temperature 55°C Time 30 seconds

Extension Temperature 72°C Time 15 seconds/kb of plasmid length


Final Extension 72°C Time 10 Minuts


Adapted from

Finnzymes Phusion® High-Fidelity DNA Polymerase instruction guide


PCR purification

When you have added the prefix and suffix you need to purify you BioBrick. You do this by using PCR purification.

Ingredients

Procedure

1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.

2. If pH indicator I has beein added to Buffer PB, check that the color of the mixture is yellow.

3. Place a QIAquick spin column in a provided 2 ml collection tube.

4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.

5. Discard flow-through. Place the QIAquick column back into the same tube.

6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.

7. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min.

8. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.

9. To elute DNA, add 50 μl Buffer EB (10 mM Tris•Cl, pH 8.5) to the center of the QIAquick membrane wait 1 min and centrifuge the column for 1 min.

Double Digest

Of BioBrick with prefix and suffux in order to ligate it with a plasmid. We have used two different approaches of the double digest. The differences are specified below as iGEM or Kenneth.


Ingredients

iGEM: 500 ng DNA / Kenneth: 700ng DNA

Water until 42,5 μl (43 ul if BSA is excluded)

5 μl NEB buffer (Decide which buffer: [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp])

0,5 μl BSA (Not necessary if the enzymes are HF)

1 μl Restrictionsenzyme A (Decide which restriction enzymes: [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf])

1 μl Restrictionsenzyme B


Mix by flicking

Incubate for 30 min in 37°C

iGEM: Deactivate the restriction enzymes by incubating at 80 degrees in 20 min.

Kenneth: The restriction enzymes are deactivated and excluded when you purify the samples by gel electroforesis followed by gel extraction. This approach exclude the restrictionenzymes and other contaminations, but has aswell a down side - you loose some of the DNA.


Freeze until you need them. The BioBrick is now ready for ligation.


Adapted from

The BioBrick™ Assembly Manual from NEB and Ginkgo BioWorks http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf

Fourth Week

Gel Extraction Protocol



Gel Extraction Protocol


You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.


Digestion of Linearized Plasmid Backbones

This protocol is intended only for preparation of the plasmid backbones (and NOT the expression vectors), before you can ligate it with your BioBrick.


25 uL Enzyme Master Mix

5 uL NEB Buffer 2

0,5 uL BSA

0,5 uL EcoRI

0,5 uL PstI

0,5 uL DpnI

18 uL H2O


iGEM: Deactivate the restriction enzymes by incubating at 80 degrees in 20 min. You now have a plsmid concentration of 12,5 ng/uL Kenneth: Purify the plasmids by gel electroforesis followed by gel extraction.


Ligation Protocol

How to ligate a BioBrick into a plasmid backbone. Use 3 times insert (BioBrick) to 1 time plasmid (3:1) in molar amounts. In calculating the amounts of DNA consider the length of the BioBrick and the plasmid. Use 25-30 ng plasmid. Note: iGEM are ligating eqimolar amounts of insert and plsmid as the only one in the world.

Following the two different approaches of digestion, where you obtain different concentrations of the DNA, use either iGEM or Kenneths procedure for ligation. See below.

Example

Plasmid 3000 bp - 12,5 ng/uL -> use 2 uL for obtaining 25 ng plasmid backbone

CYP79 B1 1600 bp - 10 ng/uL -> to obtain 3:1 use 37,5 ng = 3,8 uL insert (12,5 ng insert correspond to eqimolar amount of the plasmid, because the CYP79 B1 is only half the size of the plasmid backbone.


iGEM

2 uL digested plasmid (12,5 ng/uL)

x uL digested BioBrick (10 ng/uL)

1 uL T4 Ligase buffer

0,5 uL T4 DNA Ligase

Add water uptil 10 uL


Kenneth

If you have used Kenneths digestion approach, you have to determine the concentrations of the BioBrick and plasmid by NanoDrop.


x uL gel extracted plasmid (25-30 ng)

x uL gel extracted BioBrick

2 uL T4 ligase buffer

0,5 uL T4 DNA ligase

Add water uptil 20 uL


Ligate at room temperature in 10 min.

Keep on ice until transformation.


Transformation protocol for pSB1C3

The only exeption to the procedure is an incubation step after adding the LB medium. Incubation at 1 hr. at 37 degrees while shaking.

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