Agarose Gel Electrophoresis

Materials

• Agarose
• SERVA DNA Stain Clear G
• 1x TAE-buffer
• 40mM TRIS (Tris-(hydroxymethyl)-aminomethane)
• 20mM Acetat
• 1mM EDTA (Ethylenediaminetetraacetic acid)

Procedure

• mix 1g Agarose with 100 ml 1x TAE in a flask
• microwave the mixture until Agarose is completely dissolved (mixture is clear), the cap should sit loose on top of the flask to prevent the loss of water by vaporization and pressure-build up
• add 5 ul (to 100 ml solution) of SERVA DNA Stain Clear G and swirl the flask a few times by hand / add 2 drops of EtBr per 50 ml solution
• pour the liquid agarose gel into the gel tray and wait for the gel to solidify
• insert gel tray into gel box after removing gel casting stand or tape
• remove comb
• load the DNA samples into the gel pockets using a pipette
• run the gel by connecting the voltage generator to the gel box
• set the voltage generator to 85 - 140 V

form: Thermo Fischer Scientific

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Chemical Transformation(E. coli)

Materials

• Selection plates (LBsolid + antibiotic)
• SOC/ LB liquid
• Chemo competent E. coli
• Plasmid DNA (1 pg - 100 ng)
• Icebox
• Preheated Thermobloc/ Water bath (42 °C)
• Preheated Thermoshaker (37 °C)
• Table Centrifuge for Reaction tubes (1.5 mL)
• Sterile Drigalski spatula/ Plating beads
• Pipettes and Pipet tips (1000 µL, 200 µL)

Procedure

• Thaw 1 Aliquot (50 µL) of chemo-competent E. coli cells on ice for 10 minutes. Thaw on ice until the last ice crystals disappear
• Add 1 pg - 100 ng of plasmid DNA (1 - 5 µL) to cells and mix without vortexing
• Place on ice for 30 minutes
• Heat shock at 42 °C for 45 - 60 seconds
• Place on ice for 5 minutes
• Add 950 µL of room temperature liquid SOC or LB
• Place at 37 °C for 60 minutes. Shake vigorously (250 rpm)
• Spread 200 µL on selection plates (LB solid + AB concentration) and plate with the help of a drigalski spatula/ plating beads
• Centrifuge until there is a cell pellet (ca. 1-2 min.) and discard the supernatant up to 50 µL
• Resuspend the cell pellet in the 50 µL and plate with the help of a drigalski spatula/ plating beads
• Incubate the selection plates at 37 °C for ca. 12 hours (over night)
• Count the transformed E. coli colonies and calculate the transformation efficiency with the formula (TE) = Colonies/µg/Dilution

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PCR using Q5 polymerase

Materials

• Thermocycler
• PCR tubes
• Pipette
• Q5 Polymerase
• primer
• DNA Template
• 5x Q5 reaction buffer
• dNTPs
• bidest. H₂O

Procedure

Mastermix:

Component	25 ul	Final Concentration
Q5 High-Fidelity 2X Master Mix	12,5 ul	1X
10 uM Forward Primer	1,25 ul	0,5 uM
10 uM Reverse Primer	1,25 ul	0,5 uM
Template DNA		1 pg - 1 ng
Nuclease Free Water	to 25 ul

Thermocycler Conditions:

Step	Temperature	Time
Initial Denaturation	98°C	30 sec.
Denaturation	98°C	5-10 sec.	30-35 cycles
Annealing	60°C	10-30 sec.	30-35 cycles
Elongation	72°C	30 sec./ 1kb	30-35 cycles
Final Elongation	72°C	2 minutes
Hold	4°C

from: New England Biolabs GmbH

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Plasmid Isolation Alkaline Lysis (E. coli)

Materials

• Buffer 1:
• 50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 100 mg/mL RNase A, (store at 4˚C)
• Buffer 2:
• 0.2 M NaOH; 1 % (w/v) SDS
• Buffer 3:
• 3 M Potassium acetate, pH 5.5

Procedure

• Picking of an E.coli colony and inoculation of 3-5 mL LB liquid + appropriate antibiotic into a test tube.
• incubation at 37 °C and 150 - 200 rpm overnight
• The next day, transfer 1.5 mL of each preculture into 2.0 mL of eppis
• Centrifuge for 1 -2 minutes at 13,000 rpm
• Discard supernatant and centrifuge again for 30 seconds at 13,000 rpm
• Thoroughly pipette off the entire supernatant
• Pipette 100 µL of solution 1 onto the pellet and resuspend by vortexing (Thorough).
• Incubate the eppis at room temperature for 5-7 min.
• Add 200 µL solution 2 and CAREFULLY invert (Do not vortex!) -> solution should clear up
• Incubate on ice for 2-3 minutes
• Add 150 µL solution 3 (precipitation of DNA and proteins) and invert
• Centrifuge for 15 minutes at 16,000 rpm (optimal 14 °C, RT also ok)
• Pipette supernatant carefully into a 1.5 mL eppi (do not take a pellet)
• First wash step with 100 % ethanol 1000 µL
• Invert, centrifuge at 16,000 rpm for 12 min.
• Discard supernatant and perform 2nd wash step with 70 % ethanol at 16,000 rpm for 5 minutes
• Remove supernatant (pipette off) and dry pellets
• Dissolve the pellets in 30 µL nuclease-free water (+RNase).
• Measure the DNA concentration on the NanoDrop (note c in ng/ µL on the eppi page).
• Freeze dissolved plasmid DNA

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NEBuilder Hifi NDA Assembly

	2-3 Fragment Assembly
Recommended DNA Molar Ration	vector:insert = 1:2
Total amount of fragments	0.03-0,2 moles X ul
NEBuilder Hifi DNA Assembly Master Mix	10 ul
Deionized H2O	10-X ul
Total Volume	20 ul

• Incubate samples at 50°C for 60 minutes

from: New England Biolabs GmbH

Protein Expression in strain KRX

• Grow starter cultures overnight in LB medium (with antibiotic) at 37°C and 275 rpm
• Dilute overnight starter cultures 1:100 into LB containing antibiotic
• Grow cultures at 37°C and 275 rpm until (O.D.600) of 0.4–0.5
• Shift cultures to another incubator shaker set at 15–25°C, and continue shaking at approximately 275 rpm.
• When the cultures reach an O.D.600 of 0.5–0.6 for LB, induce protein expression by adding rhamnose to a concentration of 0.1% (1:200 dilution of 20% rhamnose)
• Grow cultures overnight at 15–25°C and 275 rpm
• Harvest cells by centrifugation (e.g., 10,000 × g for 3 minutes in a microcentrifuge or 4,000 × g for 10 minutes in a larger format

from: Promega

Recipes for media/buffers/solutions

LB solid:

10 g peptone/triptone

5 g yeast extract

10 g sodium chloride

ad 1 L and autoclave

Solution 1 (alkaline lysis):

9 g glucose

25 mL 1M Tris-HCl (pH 8.0)

20 mL 0.5M EDTA (pH 8.0)

ad 1 L sterile dH2O

not autoclave!

Solution 2 (Alkaline Lysis):

100 mL SDS stock solution (1 %)

8 g sodium hydroxide

ad 1 L sterile dH2O

not autoclave!

Solution 3 (Alkaline lysis):

600 mL 5M potassium acetate

115 mL glacial acetic acid

ad 1 L sterile dH2O!

not autoclave!