The Lambda Protocol Physics Investigation

This experiment is designed to produce DNA bound in gel to fragment according to the restriction enzyme used to cut it up and to the sizes of these subsequent fragments. The aim of this experiment is to produce a DNA fragmentation, which will be of known sizes because the entirety of the DNA genome has been mapped. So running D NA alongside other DNA will allow the size of the other DNA to be found.

Prediction: As in the experiment there are restriction enzymes being used on the DNA, and then running it in gel electrophoresis, to separate the varying size DNA fragments, then it is possible to estimate the results.

The DNA, after gel electrophoresis will form bands and smears. The smears are where there are many fragments of  DNA of a wide range of sizes, and bands are numerous DNA fragments of similar size. Subsequently, as the entirety of the D NA genome has been mapped, and the restriction enzymes used have had their recognition sites identified, then the fragmentation of the DNA can be predicted.

Fragmentation of -DNA using R. E. ‘s (Restriction-Enzymes) EcoR1, BamH1 and HindIII So, from this, the bands and smears that appear could be predicted.

It would also be reasonable to show that the column with no restriction enzymes present would have no fragmentation of the DNA, as the DNA would not be broken down. For EcoRI: There will be one band near the start (21 226 base pairs fragment) as it is the biggest and so will travel the shortest in the gel electrophoresis.

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There will be another band about 2-3 times as far as the first, which is the 7 421 base pair fragment. I’m reasoning it will be about 2-3 times as far because it is around a third of the size. Next, there will be either one smear or several fragments depending on how far the remaining fragments have moved.

As they are all similar in size then they may form an indivisible smear, which has all the DNA fragments in close proximity. If the fragments move quick enough in the gel electrophoresis, and are left for long enough, then the fragments will become further apart and may form individual definable bands. 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. 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 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 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 DNA was not broken up.

This left the entire 48 502 b. p. sequence of the 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 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.