For BamHI: The fragment 16 841 b.p. will form one band near the wells. There may be one band of 7 233b.p but this may have not travelled far enough to be distinguishable from the subsequent fragments as it is similar in size to 6 527 and 6 770. There will be 2 bands, 6 527 b.p. with 6 770 b.p and of 5 505 b.p. with 5 626 b.p. These may however form one smear, as they are similar in size.
For HindIII: There will be one band near the start, closest than any fragments of the other wells of the (23 130 b.p.). There will be one band about over twice as far out (9,416). Then one band of 6 557 b.p., which may contain fragment 4 361b.p. as a smear, then one band of 2 027 and 2 322 base pairs. This band may smear with the previous smear. There will be a small band much further out compared to the rest of the fragments and the other wells consisting of fragments of 564 and 125 base pairs. This is because they are the smallest and so will travel the furthest.
The Method:
To prepare for gel electrophoresis of the λ-DNA:
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Hydrate the λ-DNA by adding distilled water (100 μl) to the dried λ-DNA. (Fig 2.) The λ-DNA solution is used to allow electricity to be more easily conducted through the λ-DNA, and to provide an aqueous solution of λ-DNA, which can flow freely in the agarose.
- Divide the DNA solution into four tubes, each with 20 μl. Each tube already contains the restriction enzymes, with the last one containing no restriction enzymes. (Fig 3.)
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Incubate these tubes for 35-40 minutes to allow the restriction enzymes to restrict (divide) the λ-DNA. (Fig 4.)
- Add molten agarose to the electrophoresis tank. (Fig 5.)
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Add 2 μl of loading dye to the enzyme-λ-DNA tubes and mix. Then add to the wells of the electrophoresis tank. Then flood the tank and wells with buffer solution (Ions in the buffer solution conduct the electricity, and prevent dehydration of the λ-DNA) (Slightly more than 10 cm3) Gently remove the dividing wall of the tank to allow the buffer solution to spread around the set agarose gel. The conductive buffer solution allows the electricity to pass through the tank via the buffer solution so a circuit can be easily made and the electrophoresis can take place. (Fig. 6)
- Place Carbon Fibre electrodes to the electrophoresis tank. Then attach crocodile clips wired to 2x 9-volt batteries. The electricity on DC is what allows the negatively charged phosphate groups (induced by the alkaline buffer solution) to move towards the positive terminal, the anode. (Fig 7.)
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Leave for 12 hours, to allow time for the migration of the λ-DNA.
- Remove the electrodes, and then flood the tank with staining solution.
- Leave for 4 minutes, to allow the DNA to be stained, and then pour off excess staining solution.
Points:
- The λ-DNA is hydrated to allow an aqueous solution to conduct electricity more easily and to allow it to migrate freely in the agarose gel. Also the restriction enzymes can access the λ-DNA easily in solution, which is during incubation. The solution must be thoroughly mixed
- The agarose gel is used as a medium for the λ-DNA solution to migrate across.
- Gloves must be worn to prevent contamination of handling the λ-DNA. If you touch the micropipette, enzymes in sweat may contaminate the λ-DNA, degrading it. (Such as DNAases and proteases.)
- Plastic tubes are used because DNA sticks to glass.
Results:
Conclusion:
For EcoRI, the number of fragments in these results was equal to the number of results foretold by the comparison of the (complete) λ-DNA genome. Between the first and second band, there was a 1 mm difference, and the remaining five bands had 2 mm difference. This is reasonable as according to the λ-DNA genome, the base pairs formed (ignoring 21 226 b.p. fragment) were about 1000 b.p difference. According to my prediction, there should be a greater gap between the first and second band, but the rest of the bands are not unexpected.
For BamHI there were 3 bands formed, 2,11 and 13mm. This is reasonable because there was one large fragment, 16 841 b.p. (the 2mm distance) and then 2 other bands, which are likely to be the 6 527 with 6 770 (b.p.) (with 7 233) and the other with 5 626 and 5 505 (b.p.)
For HindIII 4 fragments formed, 4,6,9,11 (mm). There are, however 5-6 distinguishable bands shown by the λ-DNA-restriction enzyme chart. The missing band could be explained by: the smallest fragments, 564 125 (b.p.), not showing up on the staining because they are too small. Or the biggest fragment (23 130), which is also the biggest fragment out of all the λ-DNA fragmentations (with the different R.E’s) may not have been distinguishable from the well itself. Given by the smallest distance was 4mm from the well and as this is the biggest fragment; comparing to the similar size, but smaller EcoRI fragment (21 226 b.p.) travelling 3 mm, it would say that it was this that did not move enough from the well to be identified separately from the well. Assuming this is the cause, and then the remaining fragments are not unexpected. 4mm is the 9 416 (b.p.) fragment, the 6 557 b.p. being the 6mm fragment. The remaining 9mm and 11mm are the 2 027 with 2 322 (b.p.) fragments and the 564, with 125 b.p. fragments respectively.
The results for the no restriction enzyme column were more than the prediction stated. There were four bands identified but this should have been 1 band that did not move far at all. This is because as there were no restriction enzymes present then the λ-DNA was not broken up. This left the entire 48 502 b.p. sequence of the λ-DNA genome, which should not have moved far, and not into 4 fragments.
From these results, the experiment was pretty accurate, comparing to the prediction. There were no unexpected results, except one of the fragments disappearing in HindIII, but, as stated, was probably due to it being undistinguished from the well, the source.
Evaluation:
Comparing to the prediction of ‘what should happen’, the experiment was successful. A possibly way of augmenting the experiment is leaving the current on for longer when letting the λ-DNA run in the gel electrophoresis tank. This would allow greater seperation between the bands to allow closer examination of the separate fragments. Increasing the voltage may prove to be damaging, as it may break down the DNA.
The results that were not according to the prediction were the no enzyme column. The prediction said that there should be no fragments and that the single fragment (ie. The entire genome) should have moved very little. The possibly causes of this was the DNA was damaged in the handling or that the tube was contaminated at some stage.
