Team:UQ-Australia/Notebook/Protocols

From 2011.igem.org

(Difference between revisions)
Line 22: Line 22:
Using the Phusion PCR kit:
Using the Phusion PCR kit:
 +
25ul Phusion mix
25ul Phusion mix
 +
1.25ul each primer (at 100uM)
1.25ul each primer (at 100uM)
 +
2ul DNA
2ul DNA
 +
1.5ul DMSO
1.5ul DMSO
 +
19ul water
19ul water
 +
1. 98C for 30s
1. 98C for 30s
 +
2. 98C for 10s
2. 98C for 10s
 +
3. 65C for 30s
3. 65C for 30s
 +
4. 72C for 30s
4. 72C for 30s
 +
5. 72C for 10min
5. 72C for 10min
 +
6. 12C FOREVER
6. 12C FOREVER
 +
cycle 34x between 2-4
cycle 34x between 2-4
Line 39: Line 51:
Using QIAprep Spin Miniprep kit:
Using QIAprep Spin Miniprep kit:
 +
1. Resuspend bacteria pellet in 250ul P1 buffer
1. Resuspend bacteria pellet in 250ul P1 buffer
 +
2. Add 250ul P2 buffer and mix by inverting
2. Add 250ul P2 buffer and mix by inverting
 +
3. Add 350ul N3 buffer and mix by inverting
3. Add 350ul N3 buffer and mix by inverting
 +
4. Centrifuge 10mins at max, take supernatant and pipette into QIAprep spin column
4. Centrifuge 10mins at max, take supernatant and pipette into QIAprep spin column
 +
5. Centrifuge 30-60s, discard flow through
5. Centrifuge 30-60s, discard flow through
 +
6. Add 0.75ml buffer PE and centrigue 1min, discard flow through
6. Add 0.75ml buffer PE and centrigue 1min, discard flow through
 +
7. Centrifuge an additional one min to remove residual buffer
7. Centrifuge an additional one min to remove residual buffer
 +
8. Place QIAprep column in a new tube and add 50ul buffer EB, spin for 1min
8. Place QIAprep column in a new tube and add 50ul buffer EB, spin for 1min
Line 51: Line 71:
35ul DNA
35ul DNA
 +
5ul Buffer (10x, NEB buffer 3)
5ul Buffer (10x, NEB buffer 3)
 +
0.5ul EcoRI
0.5ul EcoRI
 +
0.5ul PstI
0.5ul PstI
 +
0.5ul BSA (100x)
0.5ul BSA (100x)
 +
8.5ul water
8.5ul water
 +
Placed in 37C waterbath for 1hr
Placed in 37C waterbath for 1hr
Line 62: Line 88:
Using Zymoclean purification kit:
Using Zymoclean purification kit:
 +
1. For each volume of agarose, add 3 volumes of ADB and incubate for 10mins at 55C.  
1. For each volume of agarose, add 3 volumes of ADB and incubate for 10mins at 55C.  
-
2. Transfer melted agarose into a zymo-spin column and place in a collection tube, centrifuge at max for 1min and then discard liquid.
+
 
 +
2. Transfer melted agarose into a zymo-spin column and place in a collection tube, centrifuge at max for 1min and then  
 +
discard liquid.
 +
 
3. Add 200ul wash buffer and centrifuge at max for 30s
3. Add 200ul wash buffer and centrifuge at max for 30s
 +
4. Add 10ul RNA-free water and spin for 1min.
4. Add 10ul RNA-free water and spin for 1min.
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USing NEB Quick Ligation kit:
USing NEB Quick Ligation kit:
 +
1ul DNA ligase
1ul DNA ligase
 +
10ul Buffer (2x)
10ul Buffer (2x)
 +
Volume of insert and vector varies depending on concentration
Volume of insert and vector varies depending on concentration
 +
Brought to 20ul total volume with water.
Brought to 20ul total volume with water.
 +
Leave at room temp for 5mins then put on ice.
Leave at room temp for 5mins then put on ice.
Line 80: Line 116:
1. Thaw cells (TOP10) on ice. To 25ul of cells add 5ul of the ligation and mix gently.  
1. Thaw cells (TOP10) on ice. To 25ul of cells add 5ul of the ligation and mix gently.  
 +
2. Incubate on ice for 30mins then heat shock at 42C for 30secs.
2. Incubate on ice for 30mins then heat shock at 42C for 30secs.
 +
3. Place back on ice for 5mins before adding 1ml of pre-warmed SOC media.
3. Place back on ice for 5mins before adding 1ml of pre-warmed SOC media.
 +
4. Shake at 37C for 1hr at 225rpm.
4. Shake at 37C for 1hr at 225rpm.
 +
5. Spread 100ul onto a pre-warmed selective plate and incubate overnight at 37C.
5. Spread 100ul onto a pre-warmed selective plate and incubate overnight at 37C.

Revision as of 08:57, 4 October 2011





This page lists the protocols for all the experiments we conducted regularly. Unless otherwise noted, these are the standard procedures we used for each of the processes mentioned below. IGEM basic Logo stylized.png
UQ-Australia logo 2011.png


Glycerol Stocks

1.75mL of bacterial culture is added to 0.5ml of LB+50% glycerol.

Gel Electrophoresis

1% w/v agarose gels were used in 1% TAE Buffer. Gels were run at 70v for 30mins.

PCR

Using the Phusion PCR kit:

25ul Phusion mix

1.25ul each primer (at 100uM)

2ul DNA

1.5ul DMSO

19ul water


1. 98C for 30s

2. 98C for 10s

3. 65C for 30s

4. 72C for 30s

5. 72C for 10min

6. 12C FOREVER

cycle 34x between 2-4

Miniprep

Using QIAprep Spin Miniprep kit:

1. Resuspend bacteria pellet in 250ul P1 buffer

2. Add 250ul P2 buffer and mix by inverting

3. Add 350ul N3 buffer and mix by inverting

4. Centrifuge 10mins at max, take supernatant and pipette into QIAprep spin column

5. Centrifuge 30-60s, discard flow through

6. Add 0.75ml buffer PE and centrigue 1min, discard flow through

7. Centrifuge an additional one min to remove residual buffer

8. Place QIAprep column in a new tube and add 50ul buffer EB, spin for 1min

Restriction Digestions

35ul DNA

5ul Buffer (10x, NEB buffer 3)

0.5ul EcoRI

0.5ul PstI

0.5ul BSA (100x)

8.5ul water


Placed in 37C waterbath for 1hr

Gel Purification

Using Zymoclean purification kit:

1. For each volume of agarose, add 3 volumes of ADB and incubate for 10mins at 55C.

2. Transfer melted agarose into a zymo-spin column and place in a collection tube, centrifuge at max for 1min and then discard liquid.

3. Add 200ul wash buffer and centrifuge at max for 30s

4. Add 10ul RNA-free water and spin for 1min.

Ligation

USing NEB Quick Ligation kit:

1ul DNA ligase

10ul Buffer (2x)

Volume of insert and vector varies depending on concentration

Brought to 20ul total volume with water.


Leave at room temp for 5mins then put on ice.

Transformations

1. Thaw cells (TOP10) on ice. To 25ul of cells add 5ul of the ligation and mix gently.

2. Incubate on ice for 30mins then heat shock at 42C for 30secs.

3. Place back on ice for 5mins before adding 1ml of pre-warmed SOC media.

4. Shake at 37C for 1hr at 225rpm.

5. Spread 100ul onto a pre-warmed selective plate and incubate overnight at 37C.

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