Agarose Gel

Materials

• 1X TAE

• Agarose

• Gel Red

• DNA samples

• 6X loading dye

• Nuclease free water

Procedure

Prepare 1.2% agarose gel for small fragments and 3% agarose gel for large fragments

• Mix 50 mL 1X TAE and 0.6 g (1.2%) or 1.5 g (3%) agarose

• Melt in microwave until agarose has melted (about 50 seconds)

• Add 1.3 μL (1.2%) or 1.5 μL (3%) Gel Red

• Pour solution into agarose gel mold with comb

• Let set for 20 minutes or until solid

• Place gel in 1X TAE and remove comb

• Load samples of 200 ng (or 2 μL) DNA mixed with 2 μL 6X loading dye and nuclease free water up to 12 μL

• Run gel at 100-120 Volts for 40-50 minutes (1.2%) or 80 Volts for 2 hours (3%)

• Take a picture of the gel at the UV detector

Amino acid solution

Materials

• Histidine-Hcl

• Uracil

• Leucine

• Tryptophan

Procedure

Stock concentration	Final concentration	Total quantity for 50 mL
100 mM Histidine-Hcl (209 g/mol)	20.9 g/L	0.418 g
20 mM Uracil (112 g/mol)	2.24 g/L	0.0448 g
100 mM Leucine (131 g/mol)	13.1 g/L	0.262 g
40 mM Tryptophan (204 g/mol)	8.16 g/L	0.1632 g

• Filter and sterilize solutions

• Add 8 mL per liter of selective medium or spread 500 μL on a selective plate

Colony PCR - based on NEB Taq PCR Protocol

Materials

• 10X Standard Taq Reaction Buffer

• dNTPs

• Taq polymerase

• Forward and Reverse Primers

• Nuclease Free Water

• Petri dish with transformed colonies

Procedure

• Prepare following reaction in 0.5 mL PCR tubes on ice by making a Master Mix for all reactions and adding polymerase last:

Component	25 μL reaction	Final concentration
10X Standard Taq Reaction Buffer	2.5 μL	1X
10 mM dNTPs	0.5 μL	200 μM
10 mM Forward Primer	0.5 μL	0.2 μM
10 mM Reverse Primer	0.5 μL	0.2 μM
Taq DNA Polymerase	0.125 μL	0.625 units/25 μL PCR
Nuclease Free Water	to 25 μL

• With a sterile tip, under the flame, scrape part of a single colony and add to PCR tube

• Mix by pipetting up and down or flicking the reactions

• Put tubes in thermocycler with following cycling conditions:

Step		Temperature	Time
Initial Denaturation		95°C	30 seconds
25 – 35 cycles	Denaturation	95°C	15 – 30 seconds
	Annealing	45 – 68°C	15 - 60seconds
	Extension	68°C	1 minutes per kb
Final Extension		68°C	5 minutes
Hold		4°C

Gibson Assembly - based on NEB Gibson Assembly Protocol

Materials

• DNA fragments

• 2X Gibson Assembly Mater Mix (NEB)

• 2X NEBuilder Positive Control (NEB)

• Deionized water

Procedure

• Set up following reactions on ice, adding Gibson Assembly Master Mix last:

Component	2 – 3 Fragments Assembly	4 – 6 Fragments Assembly	Positive Control
Total Amount of Fragments	0.02 – 0.5 pmols	0.2 – 1 pmols	10 μL
2X Gibson Assembly Master Mix	10 μL	10 μL	10 μL
Deionized water	to 20 μL	to 20 μL	0 μL

Optimized efficiency for 50 – 100 ng of vectors and 2 – 3 fold of excess inserts

• Incubate samples at 50°C for 15 minutes (2 – 3 fragments) or for 60 minutes (4 – 6 fragments)

• Store samples on ice or at -20°C until transformation

• Transform competent cells following the Transformation Protocol

Miniprep - with QIAprep Spin Miniprep Kit (QIAGEN)

Materials

• Overnight liquid cell cultures (5 – 8 mL)

• QIAprep Spin Miniprep Kit

Procedure

• Centrifuge liquid cell cultures at 4000 rpm for 10 minutes

to be continued, I'm going Paleo :)