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Plasmid Isolation & Restriction Mapping (pGLO Purification…
Plasmid Isolation & Restriction Mapping
pGLO Purification
resuspend pellet in 125 ul p1 by pipetting in and out then 'pool' them in one microcentrifuge tube
Spin @ 6000rpm for 5 min and decant supernatant (bio waste beakers)
Add 1.5 ml of culture to two microcentrifuge tubes
Add 250 ul of P2 -> invert then Add 350 ul of N3 -> invert
Spin for 10 min. @ 13,000rpms and pipette supernatant out to place in ion exchange column
Spin column at 13,000 rpm for 1 min...liquid-> Waste beaker
Add 500 ul of PB -> 13,000 for 1 min -> Discard liquid Add 750 ul of PE -> 13,000 for 1 min -> Discard liquid Empty collection tube -> 13,000 for 1 min
Recover: place column in a microcentrifuge -> add 50 ul of EB - > After 1 min -> Spin 13,000 for 1 min. Discard spin column and label microcentrifuge tube
~50 + ~50 = 100 ul plasmid GLO
Restriction Mapping of pGlo
Label 1-8 microcentrifuge -> ice bucket add water, 10x buffer, EcoR1, EcoRV, Scal tubes (restriction enzymes)
Incubate for 1 min in 37 C dry bath -> recover and label
Electrophoresis Gel Preparation
Set casting tray -> pour 100 ul into casting tray of 1% argose in 1x TBE w/ EtBr 55 C
Wait -> gel set -> place in chamber -> pour 1x TBE to fill chamber and gel to depth of 1 mm
Loading Electrophoresis Gel
Add 4 ml of dye to pGLO DNA digest and mix -> spin samples
Load samples on wells ladder,1,2,3,4,5,6,7,8,ladder ladder = 6 ul 1-8 = 24 ul
Connect chamber to the voltage -> 100 V -> 1 min Turn off voltage...gel documentation?
Place gel in UV light box & see DNA...take picture
Mapping of Restriction Sites
Determine sizes of restriction fragments from the distance moved away from wells and comparing them to the ladder.
Enter your data in table
Estimate overall size of plasmid
Map the plasmid diagrammed below w/ restriction sites
