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

Grp enz 1 enz 2 NEB buffer
ASP Xho1 Hind3 2
BLW EcoRV Cla1 3, 4, (2)
CHC Bgl2 Cla1 3, 4
CNT pvu1 EcoRV 3
EFO Hpa1 Mlu1 4, 3
KSY Spe1 Sph1 2
LSO Sal1 EcoRV 3
NOD EcoRV Sac1 1, 2, 3, 4
RFR Cla1 Hind3 4,2,
RMT Bgl2 Pvu2 3

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
AttachmentSize
semi-log_paper.doc90.5 KB
2-cycle_semi-log.pdf24.72 KB