Our Protocols

From 2008.igem.org

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'''1.) Making Agarose Gel (standard 1%)'''
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[[Image:UniShefBanner.jpg|center]]
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Time: ~1 hour
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{| style="color:#888888;background-color:##888888;" cellpadding="5" cellspacing="2" border="2" bordercolor=#888888 width="85%" align="center"
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!align="center"|[[Team:University_of_Sheffield |Introduction]]
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!align="center"|[[Team:University_of_Sheffield /Project|Our project]]
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!align="center"|[[Team:University_of_Sheffield /Modelling|Modelling]]
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!align="center"|[[Team:University_of_Sheffield /Parts|Parts]]
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!align="center"|[[Team:University_of_Sheffield /Lab Books| Our Team]]
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!align="center"|[[Team:University_of_Sheffield /Calendar| Calendar]]
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|}
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Required:
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__TOC__
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=Making Agarose Gel=
 +
Standard 1%
 +
 
 +
'''Time:'''
 +
* ~1 hour
 +
 
 +
'''Required:'''
*Gel tank, with combs, casting tray and relevant power supply
*Gel tank, with combs, casting tray and relevant power supply
*Agarose powder
*Agarose powder
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''Method''
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'''Method'''
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1. The gel tank we normally use holds 150ml, therefore, take 150ml of 1x TAE Buffer
+
1.) The gel tank we normally use holds 150ml, therefore, take 150ml of 1x TAE Buffer
-
2. The gel is supposed to be 10% agarose, therefore we need to add 1.5g agarose powder
+
2.) The gel is supposed to be 10% agarose, therefore we need to add 1.5g agarose powder
-
3. Combine the two in a chemical screw-cap bottle, and, leaving the lid slightly loose
+
3.) Combine the two in a chemical screw-cap bottle, and, leaving the lid slightly loose
-
4. Cook on high heat in the microwave for 2 mins, or until ALL the agarose has dissolved (it may stick to the bottom, swirling may be required. *CAUTION* very hot liquid)
+
4.) Cook on high heat in the microwave for 2 mins, or until ALL the agarose has dissolved (it may stick to the bottom, swirling may be required. *CAUTION* very hot liquid)
-
5. If it starts to overboil, pause the microwave, allow to calm down, and continue.
+
5.) If it starts to overboil, pause the microwave, allow to calm down, and continue.
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6. When all the agarose has dissolved, remove carefully! Its very hot! Oven gloves may be required.
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6.) When all the agarose has dissolved, remove carefully! Its very hot! Oven gloves may be required.
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7. Add 10 µl of ethidium bromide per 100ml of buffer. Swirl to mix. Warning! Ethidium bromide is carcinogenic and therefore very dangerous!
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7.) Add 10 µl of ethidium bromide per 100ml of buffer. Swirl to mix. Warning! Ethidium bromide is carcinogenic and therefore very dangerous!
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8. Leave until touchably cool before pouring into gel tank. Gently bathe bottle in cold water to speed up the process if necessary.
+
8.) Leave until touchably cool before pouring into gel tank. Gently bathe bottle in cold water to speed up the process if necessary.
-
9. Make sure combs are in place in tank, and casting tray is secured on all sides, then pour in the liquid.
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9.) Make sure combs are in place in tank, and casting tray is secured on all sides, then pour in the liquid.
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10. Leave until cool (~30 mins)
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10.) Leave until cool (~30 mins)
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----
 
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'''2.) PCR Purification (Using QIAGEN Kit)'''
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=PCR Purification=
 +
Using QIAGEN Kit
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Time: ~1 hour
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'''Time:'''
 +
* ~1 hour
-
Required:
+
'''Required:'''
-
*PCR Purification kit ideally. Instructions and reagents usually provided
+
* PCR Purification kit ideally. Instructions and reagents usually provided
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''Method''
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'''Method'''
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1. Add 5 volumes of PBI buffer (provided) for every 1 volume of PCR product in an eppendorf tube. Make sure one eppendorf is large enough – you may need more.
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1.) Add 5 volumes of PBI buffer (provided) for every 1 volume of PCR product in an eppendorf tube. Make sure one eppendorf is large enough – you may need more.
-
2. Put mixture in a QIAGEN column (purple) and into a 2ml collection tube (clear)(both provided). Remember, these columns only hold 700µl! Again you may need to repeat steps 3 and 4 more than once.
+
2.) Put mixture in a QIAGEN column (purple) and into a 2ml collection tube (clear)(both provided). Remember, these columns only hold 700µl! Again you may need to repeat steps 3 and 4 more than once.
-
3. Centrifuge at 10000-13000g for 30-60seconds
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3.) Centrifuge at 10000-13000g for 30-60seconds
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4. ‘Flow through’ will be found in the collection tube: discard. The DNA has stuck to the column membrane. Repeat if you had more than 700µl of PCR+PBI buffer to start with
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4.) ‘Flow through’ will be found in the collection tube: discard. The DNA has stuck to the column membrane. Repeat if you had more than 700µl of PCR+PBI buffer to start with
-
5. The wash DNA of impurities further, add 0.75ml (750µl) of PE buffer (provided)(make sure the ethanol HAS been added to it! You have to do this yourself, but previous users of the kit may have done so and should have labelled the bottle so).
+
5.) The wash DNA of impurities further, add 0.75ml (750µl) of PE buffer (provided)(make sure the ethanol HAS been added to it! You have to do this yourself, but previous users of the kit may have done so and should have labelled the bottle so).
-
6. Centrifuge for 1 min, and discard flow through.
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6.) Centrifuge for 1 min, and discard flow through.
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7. Centrifuge for a further 1min, as not all the PE buffer from the last step could fit in the collection tube.
+
7.) Centrifuge for a further 1min, as not all the PE buffer from the last step could fit in the collection tube.
-
8. Discard flow through and put column into where you want your eluate (usually 1.5ml eppendorfs)
+
8.) Discard flow through and put column into where you want your eluate (usually 1.5ml eppendorfs)
-
9. To elute DNA, add 50µl of EB buffer (provided). This ‘unsticks’ the DNA allowing it to be washed through.
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9.) To elute DNA, add 50µl of EB buffer (provided). This ‘unsticks’ the DNA allowing it to be washed through.
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10. Centrifuge for 1 min
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10.) Centrifuge for 1 min
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11. KEEP the flow through! This has your DNA in.
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11.) KEEP the flow through! This has your DNA in.
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----
 
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'''1.) Making SOB Medium'''
 
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Time: ~20 mins, plus autoclaving time
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=Making SOB Medium=
-
Required:
+
 
 +
'''Time:'''
 +
* ~20 mins, plus autoclaving time
 +
 
 +
'''Required:'''
EITHER
EITHER
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''Method''
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'''Method'''
1.) Combine dH2O, tryptone, yeats and NaCl, and shake until combined
1.) Combine dH2O, tryptone, yeats and NaCl, and shake until combined
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----
 
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'''4.) Making SOC Medium'''
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=Making SOC Medium=
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''Method''
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'''Method'''
Same as for SOB above, but add 20mM glucose
Same as for SOB above, but add 20mM glucose
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----
 
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'''4.) Making Cells Electrocompetant'''
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=Making Cells Electrocompetant=
 +
by dr Graham Stafford
 +
This protocol is used for making electrocompetent E.coli strain MG1655 bearing plasmid pKD46
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Time:
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'''Time:'''
 +
* ~ 3 hours cell growth
 +
* ~ 1hour 30mins execution
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Required:
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'''Required:'''
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*
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* LB Medium
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* Ice cold dH20
 +
* Ice cold 1.5ml eppendorfs
 +
* Ice cold 10% glycerol
 +
'''Method'''
 +
Centrifuge steps are 1000g for 15 mins
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''Method''
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1.) Grow E.coli strain in LB until OD 600 = 0.4
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-
----
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'''5.) Electroporation'''
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2.) Centrifuge at 4 degrees C
 +
3.) Pour of supernatant resuspend in 100 ml ice cold H2O
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Time:
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4.) Centrifuge at 4 degrees C
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Required:
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5.) Pour off supernatant, resuspend in 50 ml ice cold H2O
 +
 
 +
6.) Centrifuge at 4 degrees C
 +
 
 +
7.) Pour off supernatant, resuspend in 50ml ice cold 10% glycerol (sterile)
 +
 
 +
8.) Centrifuge at 4 degrees C
 +
 
 +
9.) Pour off supernatant, resuspend in 500ul ice cold 10% glycerol. Note: work only
 +
with freschly incubated cultures, as cultures old show no results
 +
 
 +
10.) Aliquot in 40ul portions into sterile, pre-chilled eppendorfs
 +
 
 +
11.) These can be stored at - 80 for months / years
 +
 
 +
=Electroporation=
 +
 
 +
 
 +
'''Time:'''
 +
* ~
 +
 
 +
'''Required:'''
*
*
-
''Method''
+
'''Method'''
   
   
-
----
 
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'''6.) Making Cells Chemically Competant''
+
 
 +
=Making Cells Chemically Competant=
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Time:
+
'''Time:'''
 +
* ~
-
Required:
+
'''Required:'''
*
*
-
''Method''
+
'''Method'''

Latest revision as of 22:42, 27 October 2008

UniShefBanner.jpg


Introduction Our project Modelling Parts Our Team Calendar


Contents


Making Agarose Gel

Standard 1%

Time:

  • ~1 hour

Required:

  • Gel tank, with combs, casting tray and relevant power supply
  • Agarose powder
  • TAE buffer, 1x
  • Ethidium bromide


Method

1.) The gel tank we normally use holds 150ml, therefore, take 150ml of 1x TAE Buffer

2.) The gel is supposed to be 10% agarose, therefore we need to add 1.5g agarose powder

3.) Combine the two in a chemical screw-cap bottle, and, leaving the lid slightly loose

4.) Cook on high heat in the microwave for 2 mins, or until ALL the agarose has dissolved (it may stick to the bottom, swirling may be required. *CAUTION* very hot liquid)

5.) If it starts to overboil, pause the microwave, allow to calm down, and continue.

6.) When all the agarose has dissolved, remove carefully! Its very hot! Oven gloves may be required.

7.) Add 10 µl of ethidium bromide per 100ml of buffer. Swirl to mix. Warning! Ethidium bromide is carcinogenic and therefore very dangerous!

8.) Leave until touchably cool before pouring into gel tank. Gently bathe bottle in cold water to speed up the process if necessary.

9.) Make sure combs are in place in tank, and casting tray is secured on all sides, then pour in the liquid.

10.) Leave until cool (~30 mins)


PCR Purification

Using QIAGEN Kit


Time:

  • ~1 hour

Required:

  • PCR Purification kit ideally. Instructions and reagents usually provided


Method

1.) Add 5 volumes of PBI buffer (provided) for every 1 volume of PCR product in an eppendorf tube. Make sure one eppendorf is large enough – you may need more.

2.) Put mixture in a QIAGEN column (purple) and into a 2ml collection tube (clear)(both provided). Remember, these columns only hold 700µl! Again you may need to repeat steps 3 and 4 more than once.

3.) Centrifuge at 10000-13000g for 30-60seconds

4.) ‘Flow through’ will be found in the collection tube: discard. The DNA has stuck to the column membrane. Repeat if you had more than 700µl of PCR+PBI buffer to start with

5.) The wash DNA of impurities further, add 0.75ml (750µl) of PE buffer (provided)(make sure the ethanol HAS been added to it! You have to do this yourself, but previous users of the kit may have done so and should have labelled the bottle so).

6.) Centrifuge for 1 min, and discard flow through.

7.) Centrifuge for a further 1min, as not all the PE buffer from the last step could fit in the collection tube.

8.) Discard flow through and put column into where you want your eluate (usually 1.5ml eppendorfs)

9.) To elute DNA, add 50µl of EB buffer (provided). This ‘unsticks’ the DNA allowing it to be washed through.

10.) Centrifuge for 1 min

11.) KEEP the flow through! This has your DNA in.


Making SOB Medium

Time:

  • ~20 mins, plus autoclaving time

Required:

EITHER

  • SOB powder, in which case make up as instructions

OR, per litre

  • 950ml dH20
  • 20g Tryptone
  • 5g Yeast Extract
  • 0.5g NaCl
  • 10ml of 250nM KCl
  • 5ml of 2M MgCl2
  • ~0.2ml 5M NaOH (may be required to adjust PH)


Method

1.) Combine dH2O, tryptone, yeats and NaCl, and shake until combined

2.) Add KCl

3.) Adjust to PH 7.0 with the NaOH

4.) Autoclave (20 mins liquid cycle)

5.) Add the MgCl2 just before use


Making SOC Medium

Method

Same as for SOB above, but add 20mM glucose


Making Cells Electrocompetant

by dr Graham Stafford

This protocol is used for making electrocompetent E.coli strain MG1655 bearing plasmid pKD46

Time:

  • ~ 3 hours cell growth
  • ~ 1hour 30mins execution

Required:

  • LB Medium
  • Ice cold dH20
  • Ice cold 1.5ml eppendorfs
  • Ice cold 10% glycerol

Method Centrifuge steps are 1000g for 15 mins

1.) Grow E.coli strain in LB until OD 600 = 0.4

2.) Centrifuge at 4 degrees C

3.) Pour of supernatant resuspend in 100 ml ice cold H2O

4.) Centrifuge at 4 degrees C

5.) Pour off supernatant, resuspend in 50 ml ice cold H2O

6.) Centrifuge at 4 degrees C

7.) Pour off supernatant, resuspend in 50ml ice cold 10% glycerol (sterile)

8.) Centrifuge at 4 degrees C

9.) Pour off supernatant, resuspend in 500ul ice cold 10% glycerol. Note: work only with freschly incubated cultures, as cultures old show no results

10.) Aliquot in 40ul portions into sterile, pre-chilled eppendorfs

11.) These can be stored at - 80 for months / years

Electroporation

Time:

  • ~

Required:


Method


Making Cells Chemically Competant

Time:

  • ~

Required:


Method

Retrieved from "http://2008.igem.org/Our_Protocols"