"

From 2012.igem.org

Protocols

Contents 1 Protocols 1.1 Chemo-competent cells 1.2 pGEM Ligation 1.3 Ligation 1.4 Chemotransformation 1.5 Restriction digest 1.5.1 control digest 1.5.2 preparative double digest 1.5.3 plasmid linearization 1.6 PCR 1.7 Gel electrophoresis 1.8 LB medium 1.9 SOB medium 1.10 Genaxxon Plasmid DNA Purification Mini Prep Kit 1.11 Genaxxon Gel Extraction Mini Prep Kit 1.12 Genaxxon PCR DNA Purification Mini Prep Kit 1.13 QIAGEN Plasmid Midi Kit

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

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

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]