Misc Staub lab protocols (compiled by Matt Robbins)

Last updated 4/18/06

Promega Lambda DNA/EcoR I + Hind III Markers

cat#: G1731

Preparation:

200 ul lambda product (the entire tube)

800 ul TE

200 ul 6X dye (provided with the lambda DNA)

1200 ul total

load 8 ul per lane

Promega 100 bp ladder

cat# G2101

250 ul ladder (entire tube)

250 ul TE

100 ul dye

600 ul total

load 12 ul per lane

dNTP's

1mM

Source:

            Promega cat# U1240 set of all four dNTP's each at 40 uM (400 ul of 100mM)

Preparation:

Add all 400 ul of each dNTP to 38.4 mls MQ or HPLC water to make 1 mM of each dNTP

1.25 mM

Source:

            Promega cat# U1240 set of all four dNTP's each at 40 uM (400 ul of 100mM)

Preparation:

Add all 400 ul of each dNTP to 30.4 mls MQ or HPLC water to make 1.25mM of each dNTP in 32 total mls

Create Exosap-it from Exonuclease I and Shrimp Alkaline Phosphatase

Combine:

500 ul SAP (Shrimp Alkaline Phosphatase , USB cat# 70092X 5000 units, 1u/ul)

50 ul of ExoI (Exonuclease I, Fermentas Inc. cat# EN0581 4000 units, 20u/ul)

Add 0.8 ul of EXOSAP to 10 ul of PCR product