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Lane – sample 1 2 3 4 5 6 7 8 Description Undigested Plasmid HindIII-digested plasmid HindIII-digested phage λ DNA Ligated phage λ DNA Undigested Plasmid with insert HindIII-digested plasmid HindIII-digested phage λ DNA (marker Ligated phage λ DNA Section A 1 2 3 4 5 6 7 8 The motility of DNA fragments in the gel is often influenced by the spatial conformation of the DNA molecule. the double helix reverts . double strands of the circular supercoiled DNA fragment when broken due to nick or cut. appearing in three forms: supercoiled or covalently closed circular DNA (ccc). the DNA fragments become smaller in size and hence experience less frictional resistance from the gel. and linear DNA. Due to its supercoiled nature. nicked (or open) circular DNA (oc). This results in the migration of this conformation of DNA to be faster than other conformations. However.
(iv) Size of your plasmid (use of m. for simplicity base pairs are always substituted to the m.e. The samples performed by this group are present from well 1-4 and the samples running from wells 5-8 are the one treated by the other collaborating group. lanes 1-4) showing would be. Below is an estimation made in bare-eye of what the fragment(s) size of the samples (belonging to the group treating the extract with no insert. Based on the result achieved and on bare-eye estimation (as indicated in the fragment determination handout): Table 1 Estimation of plasmid and λ DNA fragment size. This conformational change results in the increase in size of the DNA molecule and hence it experiences more frictional resistance from the gel.557 546 23. markers) The groups here performed a method of restriction with lysozyme and boiling (Method A) with on plasmid samples with no insert. Plasmid with no insert Well – Sample Fragment(s) size –up to down (bp) (1) Undigested plasmid No results (2)HindIII-digested plasmid No results (3) HindIII-digested phage λ DNA (marker) 23. this usually migrates at normal speed according to its size.027 (4) Ligated phage λ DNA (v) Size of other group plasmid Table 2 Estimation of plasmid and λ DNA fragment size (Plasmid with insert).322 2.130 9.557 4. uncoiled or relaxed state). Below is an estimation made in bare-eye of what the fragment(s) size of the samples (belonging to the group treating the extract with an insert.230 . the migration of open-circular DNA is slower than other conformations. and particularly on this case no calibration curve will be plotted as estimation by bare-eye is sufficient to our purpose. Plasmid with insert Well – Sample Fragment(s) size – up to down (bp) (5) Undigested plasmid No results (6) HindIII-digested plasmid No results (7) HindIII-digested phage λ DNA (marker) 23.to its normal (i.416 6. w.416 6. lanes 5-8) showing would be.361 2. As a result of this. W. As a result of this the plasmid DNA changes its conformation which is called as open-circular (oc) or relaxed.130 9. Aaij and Borst (1972) showed that linear DNA fragments migrate at rates that are inversely proportional to the log10 of their molecular weight. Linear fragments derive from nick or cut of both strands.
200 (vi) The results the group have in possession do not contain findings on the Undigested and digested plasmids. Also samples normally appear in accordance to the size and its special conformation. and insertion of the samples). It might also be that simply the DN have been not efficiently centrifuged to separated the DNA from the chromosomal material The dots on the gel picture (Image) proofs that the TBE buffer used to make the agarose gel was contaminated with precipitate that had been on the picture with the spots. The ligation samples were shown vivid of a widely spanned brightness. However if we were to regard the standard practical result the number of fragments obtained would be 2 in lane 2 and no results in the case of lane 6. because the cut originally present was phosphorylated by the T4 ligase resuming it to circular conformation The samples after ligation (lanes 4 and 8) show a similar migration distance thus size. circular * supercoiled * “supercoiled denatured”.361 2. On an ideal running the restricted samples should have shown evidence of cleavage by different fragments migrating at different distances from the well. The HindIII digested λ DNA. but had brightly smeared and fragments did not appear distinct in all cases (especially on lane 3). However the samples did not run as expected. digested plasmid DNA and the λ DNA restriction and ligation. Also spots appeared all through the gel. To sum the . The undigested and digested plasmids did not show on the transillumination on both groups’ lanes. Being the fragments of the digested λ DNA quite close together the gel could have been run for longer to allow a wider migration. The explanation for this appearance can be derivable from both common characteristics among the lanes (mostly related to gel running materials) and individual features (related to the preparation.027 546 23. Please see Table 1 and 2. did show positively. The sample not seen on Image could simply mean that either there was not enough DNA material or that the process treating the sample (the lysozyme and boiling method) did not work appropriately to express the restriction. due to the bright visibility of the fragments to have been well intercalated. Therefore: Brightness and smearing of sample fragments: the wells might have either been overloaded with or there must have been water or wrong concentration of the TBE buffer solution that allows the right pH level for the salts. (viii) What happened to lambda after ligation On samples running on lanes 4 and 8 a ligation was performed. if present. in the order from the well should be nicked open-circular * linear * relaxed.(8) Ligated phage λ DNA 4. The practical performed should have brought the result of the transillumination of the eight lanes running undigested vs. The original 8 fragments of DNA appeared to be clustered together due to ligation.322 2. Probably the lysozyme able alone to deteriorate the structure of a cell wall or a polymer combined with the possible alteration of the boiling could have caused denaturation of the DNA and loss of the essential elements for running. The dye ethidium bromide seems. used as a marker. that.
We will use to represent a HindIII cleavage and to represent a PstI cut.9 kb and 2. However the circular will be here adopted to resolve some hypothesis while the linear image will be used only when the order that the fragments should have will be known. Origin The site on the left is the origin Notice it would be vain to consider the arrangement of the PstI cut fragments on a circular fragment because the order would still be depending on where the origin of the Plasmid would be.1kb.1kb 2. But more importantly it is known that one of the restriction sites is the origin of the plamid. The order of the sequence of fragments can be raised when the origin of the plasmid is known. Here we analyse the different factors singularly to then bring up the resulting outcome.9kb 2.9kb The origin corresponds to the site on the right O 2. Origin 2. Before considering the final map it is necessary to consider the different factors that constitute the information of the plasmid cleavage. First factor: Possible origin of the plamid We know that HindIII cuts the plasmid into two fragments: 2.1kb O. So it is either: O The O.Section B Restriction enzyme work (analytical) (a) (i) The recognition sites can be identified in accordance to how many cleavages the enzymes have made therefore the plasmid appeared to possess 2 recognition sites for HindIII (ii) and 3 for PstI (b) The map of double digested plasmid can be displayed on a circular or linear map. Second factor: relative cleavage of PstI one with respect to HindIII (or viceversa) .
7kb. undergoing all the cuts separately (note the number of cuts in a circular plasmid always equals the number of fragments formed) o When considering from which segments the resulting fragments where formed: The 2.8kb which means that: o The cleavages formed by each of the restriction enzyme did not coincide with one another.1kb fragment (equal in length to 0. however we don’t know in what sequenced order the fragment followed as a result of the cleavage.5kb fragments) must have received ONE additional cut from HindIII to be split into two segments. Let’s eliminate all the possible chances We consider the image below where the restriction sites are at the distance suggested from the resulting fragments. Image Plasmid Map of double digestion (HindIII and PstI) . the change of position of the restriction site of one the enzymes (say PstI) would appear as a rotation as each cut should follow the rotation of one.4kb.1kb fragment received respectively two and one additional cut from PstI. We know that the 2. the other 2.5kb fragments) must have received ONE additional cut to be split into two fragments.4kb. 0. Or (which is the same) the 2.9kb fragment from HindIII (in length equal to the 0.6kb fragments) must have received another ONE additional cut by the same to be split into two.4kb fragments The final fragments resulting from the double digestion are FIVE of size: 0.8kb + 0.7kb + 1. 0. 1.Another factor to be considered from the information given is that: PstI cuts in three different sites of the plasmid forming: a 0. while the 0. Given that the cuts from a same enzyme should be rigid to the distance relative to each other.8kb segments) must have received TWO additional cuts to be split into three and the other 2.9kb fragment and the 2.7kb + 1.2kb fragment from PstI (in length equal to the 0.6kb.4 fragment remained intact In the map to be drawn we will imagine indiscriminately that the enzyme first to have digested the plasmid had been HindIII.2kb and a 2.4kb + 0.4kb fragment (equal in length to the 1. a 2.6kb + 1.5kb and 1. being PstI the second to additionally cut the fragments from HindIII Third factor: order of sub-fragments within HindIII fragments Last factor to be considered is the order in which the final segments where presented.
it is then possible to create a linear map out of the circular and consider the circumstances possible with respect to the origin location (First factor).Considering the 2.7kb (0.6kb+1.8kb one. we notice that rotating the other PstI cut within the 2.1kb) and a 0.6kb B. Origin on the right-hand HindIII restriction site .9kb fragment and counting the fragments from the HindIII cut on the left clockwise.4kb): Cannot form 0.4kb 0.1kb anticlockwise rotation. Final stage: Plasmid map Known the order of the fragments relative to one another. Counting clockwise… Image (a) and (b) Final maps of the plasmid based on the double digestion – estimation of possible arrangement of fragments A.4kb fragments not provided as resulting fragments of the double digestion.7kb fragment with the 1.7kb 1.8kb 0.4kb fragment as first or last (third within the 2. Origin on the left-hand HindIII restriction site O 1. the two PstI cuts (distant 0. from Image) which is of a 1.5kb 0.9kb fragment) because as mentioned above the cuts are distinct and cannot coincide with one another In addition considering the rotation possible to form another arrangement (equal to swapping the 0.1kb fragment would result to cleaving the latter fragment into a 1.
An agarose gel in particular is made of a complex network of polymeric molecules forming pores all through the matrix.html http://www. http://hg.askabiologist. Once the result is captured for on Polaroid of the transilluminator the visualization of the fragments process will depend on the visibility of an intercalating dye ethidium bromide that binds to the double-helices to be fluorescent under enclosed UV light. Oxford: Blackwell.wustl.php?id=6797 . Oxford: Blackwell Brown T. Section C Polyacrylamide or agarose (such as in this case) gel electrophoresis are used because suitable for the separation of polymers such as DNA molecules because of the composition of the gel through which samples are run.7kb To decide which possibility is correct it would be necessary to perform an addition of probes for each fragment and identify the result by blotting. (2001). All goes without saying that when the gel apparatus is electrically run the DNA will tend to move towards the positive pole due to its phosphate composition. Oxford: Blackwell Primrose S.8kb 0. (2006).1. B.edu/hbooks/genetics/biotech/gels/agardna.org. the larger the pores the bigger the DNA fragments that can be separated. and Twyman R. supercoiled).5kb 0. Gene Cloning & DNA Analysis Fifth Edition.6kb 1. Principles of Gene Manipulation and Genomics Seventh Edition. A.4kb 0.edu/hdk_lab_manual/plasmid/plsmid10.uk/answers/viewtopic. linear. M.vivo. The size of these pores is dependent of the agarose gel concentration and the buffer composition. The reptation of the molecule (by stretching in the direction of the applied field and contracting other) and its elastic properties through the pore of the gel matrix is as mentioned dependent to the molecular weight by an inverse proportionality and to its conformations resumed when reptating (nick-circular. References Brown T.html http://www. A. Gene Cloning & DNA Analysis Fourth Edition. (2006)..colostate.
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