Checking the size of created DNA
The DNA samples prepared yesterday today will be tested for size with electrophoreses.
Restriction Enzyme Digest
- 3μL from each DNA mini prep
- 1μL Enzym I (EcoR I)
- 1μL Enzym II (Pst I)
- 1.5μL 10x Buffer solution (enough for 1x of total volume(15μL))
- 8.5μL dH2O (enough to get total volume 15μL)
- One 0.5mL tube per sample
- Create enzyme solution.
- Add chemicals together (except DNA) into master mix
- Add dH2O first
- Add 12μL (15μL-3μL of DNA) to labeled 0.5mL tube.
- Add 3μL of DNA
- Put tubes into hotplate for 10 minutes at 37°C
While this was running an agarose gel was created.
- Assemble agarose tray.
- Slide tray into holder ensuring that gasket is in place and flush with bottom.
- Insert teeth
- Small teeth, thin up to 15μL thick 20μL
- Create agarose solution
- Locate agarose flask and fill to 60mL with agarose solution.
- Add agarose gelling powder to make 1% solution. (0.1g per 10mL)
- Swirl to mix.
- Microwave for 40s, and swirl mixture until fully dissolved.
- Microwave for 40S.
- Will be hot, may boil over if handled immediately.
- Swirl mixture intill no gels/solids are visible.
- Add 6μL of Ethidium Bromide (1μL per 10mL).
- Carcinogen avoid skin contact.
- Pour solution into tray.
- Allow to solidified for 15min.
- Carefully remove teeth and tray.
- Insert tray into electrophoresis shell
- Fill shell to Fill line.
- Fill lane one with "chemical ruler"
- Add sample to rows and record the position
- Lane 1 = Ruler
- Lane 2 = Sample 1
- Lane 3 = Sample 2
- Lane 4 = Sample 3
- Attach lid and probes
- Set voltage (110 V)
- turn on machine and watch check on gel to ensure it does not flow over.
While the gel is going the expected size can be calculated.
Find the size of expected part and plasmid back bone and compare to chemical ruler.
Gel was removed form case and placed on UV viewing stand, and a picture was taken.
| Reagent || Volume
1. 2079bp & 714bp
2. 3385bp & 2079bp
3. 2079bp & 869bp
All have PSb1A2 backbone = 2079
15 μL/lane; 1% agarose
| DNA(plasmid) || 3.0 μL
| 10X buffer || 1.5
| EcoRI || 1.0
| PstI || 1.0
| dH2O || 8.5
Electrophoresis gel under UV light.
1. >3,000 >300
2. >3,000 >2,000
3. >3,000 <1,000
These results confirm sample 2 and make probable sample 3, and sample 1 does not appear to be correct.