Team:Tuebingen/NotebookProtocols

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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
  1. 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
  1. 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

Solve agarose in TAE 1x buffer and boil until solution is clear.


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

  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]