2.5 Restriction Mapping

W 2/22/12

Goal for this lab: Purify the amplified DNA from first lab, digest it with restriction enzymes, and assess the results with gel electrophoresis.

Zymo Research DNA Clean & Concentrator-5 Cat # T1225 K

$69 for kit of 50

Reagents Per Pair:

• 125 µL Zymo-SpinTM DNA Binding Buffer
• 500 µL Zymo-SpinTM Wash Buffer (GET MORE FOR 2015)
• 1 mL Sterile ddH2O
• 1 Zymo-Spin™ Column
• 2 Capless collection tubes

• 10 µL 20 mM Spermidine [Should this be 3 tubes of 1 uL??]

• 1x TAE for 14 gels @ 300 mL/gel = 4200 mL (make at least 5 L)

• 1 2% gel per pair plus 2
• 10x NEB restriction buffer. Students need at most 10µL of any 1 buffer. Check supplies
• Fermentas MassRuler, Low Range

Restriction Enzymes for 2012

Bgl2 Cla1 EcoRV Hind3 Hpa1 Mlu1 pvu1 Pvu2 Sac1 Sal1 Spe1 Sph1 Xho1

Equipment

• Incubator at 37C, with microtube racks inside
• Water bath at 65C for gels
• copies of semi-log paper (1 per student) (see attached document)
• find powerpoint of semi-log paper and put it on the course website