Information on Running the Final pBR322 Gel

from Dr. Dellis' Lab Notebook


prepare pBR322 digests:
TUBE 0.05 ug/ul 
pBR322		 10X BUFFER EcoRI HincII PvuII# dH2O
E 5 ul 2 ul H 1 ul 12 ul
H 8 ul 2 ul B 1 ul 9 ul
P 5 ul 2 ul B 0.5 ul 12.5 ul
EH 9 ul 2 ul MC* 1 ul 1 ul 7 ul
EP 7 ul 2 ul MC 1 ul 0.5 ul 9.5 ul
HP 9 ul 2 ul B 1 ul 0.5 ul 7.5 ul
EHP 9 ul 2 ul MC
2 ul of
"EHP"
 mix 7 ul
* MC = Promega's Multicore Buffer
# only 0.5 ul of Pvu II was used to reduce star activity
 

prepare Lambda digests:
TUBE 0.1 ug/ul 
Lambda		 10X BUFFER ENZYME dH2O
C 6 ul 2 ul C 1 ul Cla I 11 ul
M 6 ul 2 ul D 1 ul Mlu I 11 ul
P 6 ul 2 ul B 1 ul Pvu II 11 ul
S 6 ul 2 ul F 1 ul Sty I 11 ul
B+C 6 ul 2 ul B 1 ul Bgl II+1 ul Csp 45I 10 ul
B+M 6 ul 2 ul D 1 ul Bgl II+1 ul Mlu I 10 ul
B+St 6 ul 2 ul B 1 ul Bgl II+1 ul Stu I 10 ul
M+Sp 6 ul 2 ul D 1 ul Mlu I+1 ul Sph I 10 ul

37'C incubation for 65 minutes

prepared 1.0% agarose midigel (22 lanes)

  • 1 gram agarose powder in 100 ml 1X TBE Buffer
  • prepared gel as usual
    • prepared two Lambda Hind III digest control lanes after gel had solidified, assembled electrophoresis apparatus following usual procedure. Added fresh 1X TBE to just cover wells.

    added 4 ul 6x TD to digest tubes and loaded gel. Order of loading:
     
    Well#1 2 3 4 5 6 7 8 9 10 11
    empty empty HindIII ClaI MluI PvuII StyI E H P EH
    12 13 14 15 16 17 18 19 20 21 22
    EP HP EHP B+C B+M B+St M+Sp HindIII empty empty empty

    EC power supply 70V run 2 hr 20 min, 12:15-2:35. Bromophenol blue band was 75~78 mm from the well (the smallest pBR322 fragment runs just behind the b.b. dye band).