2.5 Restriction Mapping

2.5 Restriction Mapping margaret Wed, 11/16/2011 - 18:16

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
semi-log_paper.doc
2-cycle_semi-log.pdf

2014 RE

2014 RE kdorfman Wed, 02/19/2014 - 20:04
MW TuTh buf1 buf 2 buf 3 buf 4 cutsmart
AatII Aat II 0 50 50 100 100
AclI AclI 100
AflII AflII 50 100 25 100 100
BciVI 100
Bgl2 10 75 100 10 10
BmgBI 3.1-100 10
BspE1 0 10 100 0 10
BsrBI 100
BsrGI 25
ClaI 10 50 50 100 100
DraI 100
EarI 100
EcoRI 100 100 100 100 50
EcoRV 50 75 100 50 10
HindIII HindIII 50 100 10 50 50
HpaI 25 50 10 100 100
MluI 25 75 100 50 25
NcoI 100 100 100 100 100
Pvu2 100 100 100 100 100
SexAI 100
SpeI 75 100 25 75 100
XbaI 0 100 75 75 100