Team:Tuebingen/NotebookProtocols

From 2012.igem.org

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(Restriction digest)
 
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__TOC__
__TOC__
== Chemo-competent cells ==
== Chemo-competent cells ==
 +
'''Inoue buffer'''
 +
{| class="wikitable"
 +
|-
 +
! Component !! Volume
 +
|-
 +
| MnCl<sub>2</sub> * 2H<sub>2</sub>0 || 9.67 g
 +
|-
 +
| CaCl<sub>2</sub> * 2H<sub>2</sub>0 || 2.2 g
 +
|-
 +
| KCl || 18.65 g
 +
|-
 +
| PIPES (0.5 M, pH 6.7) || 20 ml
 +
|-
 +
| H<sub>2</sub>0 || ad 1 l
 +
|}
 +
Sterilize through filtration (0.45 µm filter) and store at -20 °C.
 +
 +
'''Cells'''
 +
# Pick an ''E. coli'' colony and inoculate 25 ml SOB.
 +
# Let bacteria grow for 8 hours at 37 °C and 250 rpm.
 +
# Inoculate three 100 ml SOB volumes with 1 ml, 2 ml and 4 ml of the prepared pre-culture.
 +
# Incubate over night at 18 - 22 °C and 200 rpm.
 +
# At OD<sub>600</sub> = 0.55, put culture for 10 min on ice.
 +
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.
 +
# Resuspend cell pellet in 30 ml 0 °C Inoue buffer.
 +
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.
 +
# Repeat the previous two steps.
 +
# Resuspend cells in 8 ml 0 °C Inoue buffer. Add 1.5 ml DMSO and incubate on ice for 10 min.
 +
# Aliquot cells à 100 µl and freeze in liquid nitrogen. Store at -80 °C.
Line 13: Line 42:
| 2X Rapid Ligation Buffer || 5 µl
| 2X Rapid Ligation Buffer || 5 µl
|-
|-
-
| pGEM vector || 0.5 µl (25ng)
+
| pGEM vector || 0.5 µl (25 ng)
|-
|-
| PCR product  || 3.5 µl
| PCR product  || 3.5 µl
Line 19: Line 48:
| T4 DNA ligase || 1 µl (3 Weiss units)
| T4 DNA ligase || 1 µl (3 Weiss units)
|}
|}
-
 
+
Mix all reagents in a 0.5 ml tube. Incubate reaction at 4 °C over night.
-
Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.
+
Line 31: Line 59:
| 10X T4 DNA Ligase Buffer || 1 µl
| 10X T4 DNA Ligase Buffer || 1 µl
|-
|-
-
| vector DNA || 1 µl (20-100 ng)
+
| vector DNA || 1 µl (20 - 100 ng)
|-
|-
| insert DNA  || 5 µl (up to 5:1 molar ratio insert to vector)
| insert DNA  || 5 µl (up to 5:1 molar ratio insert to vector)
Line 39: Line 67:
| water || 2.5 µl
| water || 2.5 µl
|}
|}
-
Mix all reagents and incubate at 22°C for 1 hour.
+
Mix all reagents and incubate at 22 °C for 1 hour.
Line 53: Line 81:
# Add plasmid DNA to cell culture.
# Add plasmid DNA to cell culture.
# Incubate for 30 min on ice.
# Incubate for 30 min on ice.
-
# Heat shock for 90 sec at 42°C.
+
# Heat shock for 90 sec at 42 °C.
# Add 900 µl LB.
# Add 900 µl LB.
-
# Let the bacteria grow at 37°C for at least 1 hour.
+
# Let the bacteria grow at 37 °C for at least 1 hour.
== Restriction digest ==
== Restriction digest ==
 +
=== control digest ===
=== control digest ===
Line 67: Line 96:
| Tango buffer 10x || 1 µl
| Tango buffer 10x || 1 µl
|-
|-
-
| XbaI (RE) || 0.5 µl
+
| XbaI (RE) || 0.5 µl (5 units)
|-
|-
-
| SpeI (RE) || 0.5 µl
+
| SpeI (RE) || 0.5 µl (5 units)
|-
|-
| DNA || 1 µl (up to 1 µg)
| DNA || 1 µl (up to 1 µg)
Line 75: Line 104:
| water || 7 µl
| water || 7 µl
|}
|}
-
# Incubate at least for 1 hour at 37°C.
+
Incubate at least for 1 hour at 37°C.
 +
 
=== preparative double digest ===
=== preparative double digest ===
 +
{| class="wikitable"
 +
|-
 +
! Component !! Volume
 +
|-
 +
| Tango buffer 10x || 10 µl
 +
|-
 +
| SpeI (RE) || 5 µl (50 units)
 +
|-
 +
| DNA || up to 30 µg
 +
|-
 +
| water || ad 150 µl
 +
|}
 +
# Incubate for 8 hours at 37 °C.
 +
# After 3 hours add 2 µl SpeI.
 +
# Add 7 µl XbaI and incubate for another 8 hours.
 +
=== plasmid linearization ===
=== plasmid linearization ===
 +
{| class="wikitable"
 +
|-
 +
! Component !! Volume
 +
|-
 +
| Tango buffer 10x || 10 µl
 +
|-
 +
| SpeI (RE) || 7 µl (70 units)
 +
|-
 +
| DNA || up to 30 µg
 +
|-
 +
| water || ad 150 µl
 +
|}
 +
Incubate for at least 8 hours at 37 °C.
 +
== PCR ==
== PCR ==
Line 100: Line 160:
| water || 36 µl
| water || 36 µl
|}
|}
 +
 +
'''PCR conditions'''
'''PCR conditions'''
Line 106: Line 168:
! Step !! Duration !! Settings
! Step !! Duration !! Settings
|-
|-
-
| 1 || 2 min || 94°C
+
| 1 || 2 min || 94 °C
|-
|-
-
| 2 || 45 sec || 94°C
+
| 2 || 45 sec || 94 °C
|-
|-
| 3 || 30 sec || gradient or annealing temperature
| 3 || 30 sec || gradient or annealing temperature
|-
|-
-
| 4 || 90 sec || 72°C
+
| 4 || 90 sec || 72 °C
|-
|-
-
| || || steps 2-4: 30 cycles
+
| || || steps 2 - 4: 30 cycles
|-
|-
-
| 5 || 7 min || 72°C
+
| 5 || 7 min || 72 °C
|-
|-
-
| 6 || (hold) || 4°C
+
| 6 || (hold) || 4 °C
|}
|}
== Gel electrophoresis ==
== Gel electrophoresis ==
 +
'''TAE buffer 50x'''
'''TAE buffer 50x'''
{| class="wikitable"
{| class="wikitable"
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|}
|}
Adjust to pH 8.5.
Adjust to pH 8.5.
 +
 +
'''Gel'''
'''Gel'''
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| TAE 1x buffer || 120 ml
| TAE 1x buffer || 120 ml
|-
|-
-
| Agarose || 1.2 g
+
| agarose || 1.2 g
 +
|-
 +
| ethidium bromide|| 1.2 µl
|}
|}
-
Solve agarose in TAE 1x buffer and boil until solution is clear.
+
# Solve agarose in TAE 1x buffer and boil until solution is clear.
 +
# Add ethidium bromide, when lukewarm.
 +
 
 +
 
'''Well loading'''
'''Well loading'''
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|}
|}
Can be scaled up linearly.
Can be scaled up linearly.
 +
== LB medium ==
== LB medium ==
-
incl. Agarplatten
+
 
 +
{| class="wikitable"
 +
|-
 +
! Component !! Volume
 +
|-
 +
| Trypton || 10 g
 +
|-
 +
| yeast extract  || 5 g
 +
|-
 +
| NaCl || 5 g
 +
|-
 +
| water || 1 l
 +
|}
 +
Adjust to pH 7.0.
 +
 
 +
 
 +
'''Agar-plates'''
 +
{| class="wikitable"
 +
|-
 +
! Component !! Volume
 +
|-
 +
| agar-agar || 16 g
 +
|-
 +
| LB buffer || 1 l
 +
|}
 +
# Solve 16 g agar-agar in 1 l LB buffer and boil until solution is clear.
 +
# If it is nearly cold pour it into petri dishes (approx. 25 ml per dish).
== SOB medium ==
== SOB medium ==
 +
{| class="wikitable"
 +
|-
 +
! Component !! Volume
 +
|-
 +
| Trypton || 20 g
 +
|-
 +
| yeast-extract  || 5 g
 +
|-
 +
| NaCl || 0.5 g
 +
|-
 +
| 250mM KCl || 10 ml
 +
|-
 +
| water MiliQ || 1 l
 +
|}
 +
# Solve the components in 1 l water.
 +
# Autoclave.
 +
# After autoclaving add 5 ml MgCl<sub>2</sub>.
 +
 +
== Genaxxon Plasmid DNA Purification Mini Prep Kit ==
== Genaxxon Plasmid DNA Purification Mini Prep Kit ==
 +
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/Plasmid-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]
 +
 +
== Genaxxon Gel Extraction Mini Prep Kit ==
== Genaxxon Gel Extraction Mini Prep Kit ==
 +
[http://www.genaxxon.com/catalogue/DNA-Purification-Kits/PCR-and-Gel-extraction-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]
 +
 +
== Genaxxon PCR DNA Purification Mini Prep Kit ==
== Genaxxon PCR DNA Purification Mini Prep Kit ==
 +
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/PCR-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]
 +
 +
-
== Qiagen Plasmid Midi Kit ==
+
== QIAGEN Plasmid Midi Kit ==
 +
[http://www.qiagen.com/products/plasmid/qiagenplasmidpurificationsystem/qiagenplasmidmidikit.aspx#Tabs=t2 Manual provided by QIAGEN]

Latest revision as of 12:46, 26 September 2012



Protocols

Contents

Chemo-competent cells

Inoue buffer

Component Volume
MnCl2 * 2H20 9.67 g
CaCl2 * 2H20 2.2 g
KCl 18.65 g
PIPES (0.5 M, pH 6.7) 20 ml
H20 ad 1 l

Sterilize through filtration (0.45 µm filter) and store at -20 °C.

Cells

  1. Pick an E. coli colony and inoculate 25 ml SOB.
  2. Let bacteria grow for 8 hours at 37 °C and 250 rpm.
  3. Inoculate three 100 ml SOB volumes with 1 ml, 2 ml and 4 ml of the prepared pre-culture.
  4. Incubate over night at 18 - 22 °C and 200 rpm.
  5. At OD600 = 0.55, put culture for 10 min on ice.
  6. Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.
  7. Resuspend cell pellet in 30 ml 0 °C Inoue buffer.
  8. Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.
  9. Repeat the previous two steps.
  10. Resuspend cells in 8 ml 0 °C Inoue buffer. Add 1.5 ml DMSO and incubate on ice for 10 min.
  11. Aliquot cells à 100 µl and freeze in liquid nitrogen. Store at -80 °C.


pGEM Ligation

Ligation for TA-cloning of PCR products

Component Volume
2X Rapid Ligation Buffer 5 µl
pGEM vector 0.5 µl (25 ng)
PCR product 3.5 µl
T4 DNA ligase 1 µl (3 Weiss units)

Mix all reagents in a 0.5 ml tube. Incubate reaction at 4 °C over night.


Ligation

Ligation for digested parts and vectors

Component Volume
10X T4 DNA Ligase Buffer 1 µl
vector DNA 1 µl (20 - 100 ng)
insert DNA 5 µl (up to 5:1 molar ratio insert to vector)
T4 DNA ligase 1 µl (1 unit)
water 2.5 µl

Mix all reagents and incubate at 22 °C for 1 hour.


Chemotransformation

Component Volume
chemo-competent E. coli 100 µl
plasmid DNA up to 10 µl (max. 1/10 of volume)
  1. Add plasmid DNA to cell culture.
  2. Incubate for 30 min on ice.
  3. Heat shock for 90 sec at 42 °C.
  4. Add 900 µl LB.
  5. Let the bacteria grow at 37 °C for at least 1 hour.


Restriction digest

control digest

Component Volume
Tango buffer 10x 1 µl
XbaI (RE) 0.5 µl (5 units)
SpeI (RE) 0.5 µl (5 units)
DNA 1 µl (up to 1 µg)
water 7 µl

Incubate at least for 1 hour at 37°C.


preparative double digest

Component Volume
Tango buffer 10x 10 µl
SpeI (RE) 5 µl (50 units)
DNA up to 30 µg
water ad 150 µl
  1. Incubate for 8 hours at 37 °C.
  2. After 3 hours add 2 µl SpeI.
  3. Add 7 µl XbaI and incubate for another 8 hours.


plasmid linearization

Component Volume
Tango buffer 10x 10 µl
SpeI (RE) 7 µl (70 units)
DNA up to 30 µg
water ad 150 µl

Incubate for at least 8 hours at 37 °C.


PCR

Component Volume
Taq/Pfu buffer 5 µl
Taq/Pfu polymerase 1 µl
primer forward 0.5 µl (100 pmol/µl)
primer reverse 0.5 µl (100 pmol/µl)
dNTPs 2.5 µl (200 µM)
template DNA 1 µl
water 36 µl


PCR conditions

Step Duration Settings
1 2 min 94 °C
2 45 sec 94 °C
3 30 sec gradient or annealing temperature
4 90 sec 72 °C
steps 2 - 4: 30 cycles
5 7 min 72 °C
6 (hold) 4 °C


Gel electrophoresis

TAE buffer 50x

Component Volume
0.05 M EDTA 18.61 g
1 M acetic acid 60.05 g
2 M Tris 242.28 g
water 1 l

Adjust to pH 8.5.


Gel

Component Volume
TAE 1x buffer 120 ml
agarose 1.2 g
ethidium bromide 1.2 µl
  1. Solve agarose in TAE 1x buffer and boil until solution is clear.
  2. Add ethidium bromide, when lukewarm.


Well loading

Component Volume
PCR product or DNA 5 µl
Loading dye 6x 1 µl

Can be scaled up linearly.


LB medium

Component Volume
Trypton 10 g
yeast extract 5 g
NaCl 5 g
water 1 l

Adjust to pH 7.0.


Agar-plates

Component Volume
agar-agar 16 g
LB buffer 1 l
  1. Solve 16 g agar-agar in 1 l LB buffer and boil until solution is clear.
  2. If it is nearly cold pour it into petri dishes (approx. 25 ml per dish).


SOB medium

Component Volume
Trypton 20 g
yeast-extract 5 g
NaCl 0.5 g
250mM KCl 10 ml
water MiliQ 1 l
  1. Solve the components in 1 l water.
  2. Autoclave.
  3. After autoclaving add 5 ml MgCl2.



Genaxxon Plasmid DNA Purification Mini Prep Kit

[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/Plasmid-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]



Genaxxon Gel Extraction Mini Prep Kit

[http://www.genaxxon.com/catalogue/DNA-Purification-Kits/PCR-and-Gel-extraction-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]



Genaxxon PCR DNA Purification Mini Prep Kit

[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/PCR-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]



QIAGEN Plasmid Midi Kit

[http://www.qiagen.com/products/plasmid/qiagenplasmidpurificationsystem/qiagenplasmidmidikit.aspx#Tabs=t2 Manual provided by QIAGEN]