Agarose gel cataphoresis is a method used in biochemistry and molecular biological science. It is used to divide DNA or RNA molecules by size. Since Deoxyribonucleic acid and RNA have negatively charge, when they go through an agarose matrix with an electric field, they will travel from cathode to anode. The shorter molecules move faster and migrate farther than longer molecules ; hence the different sizes molecules can be separate. The most of import factor which affects the migration is the length of the Deoxyribonucleic acid molecules.
A limitation enzyme, is an enzyme that cuts dual – stranded Deoxyribonucleic acid following its specific acknowledgment of short base sequences, is used to cut the DNA into little fragments. The alone acknowledgment sequences are normally tetra- or hexanucleotide palindromes with axes of dyad symmetricalness. Which means the sequence on one strand reads the same in the rearward way on the complementary strand, e.g. GTATAC and its complementary strand CATATG. Recognition sequences in DNA differ for each limitation enzyme, bring forthing differences in the length, sequence and strand orientation of the Deoxyribonucleic acid fragments.
Plasmid is an excess – chromosomal DNA molecule, which is capable of retroflexing independently of the chromosomal DNA. It is round and dual – stranded. Plasmid is normally found in bacteriums. The size of plasmid is between 1 to 200 kilobase braces.
In this experiment, a specific recombinant plasmid pBR325 was analyzed, and therefore a limitation map would be constructed.
As described in the practical manual
The exposure of the gel, consequence informations and graph showed below.
Size of 1 kilobits ladder criterion
From the exposure, four unknown plasmid fragments migrated distance could be measured and therefore the four fragments size could be found:
In lane 2: migrated distance of the fragment was 36.5mm, hence 11.00 kilobase would be the size of this fragment ;
In lane 3: migrated distance of the close fragment was 42.5mm, hence 7.08 kilobase would be the size of this fragment ; migrated distance of the farther fragment was 66mm, hence 3.98 kilobase would be the size of this fragment ;
In lane 4: migrated distance of the close fragment was 40.75mm, hence 7.94 kilobase would be the size of this fragment ; migrated distance of the farther fragment was 73mm, hence 2.82 kilobase would be the size of this fragment ;
In lane 5: migrated distance of the close fragment was 50mm, hence 6.31 kilobase would be the size of this fragment ; migrated distance of the farther fragment was 53mm, hence 5.62 kilobase would be the size of this fragment ;
The 4 unknown plasmid size and migrated distance were showed in following tabular array:
Migrated distance ( millimeter )
Size ( kilobit )
EcoRI + BamHI
EcoRI + PstI
BamHI + PstI
From the tabular array above, the size of the plasmid could be found, which was about 11 kilobase. Since the plasmid was merely cut by EcoRI + BamHI in approximately 4:8 ratio of the plasmid, therefore the limitation map for EcoRI + BamHI could be construted:
Same method was used to happen other limitation maps:
The nucleic acids migrated from cathode to anode since it had negatively charge.
The cutting ratio of three enzymes could be found by the size of fragment as consequence showed. The last lane, which was the untrimmed plasmid, was loaded, in order to compare with the EcoRI enzyme cut fragment. The migrated distance of this untrimmed plasmid was big than EcoRI cut ‘s migrated distance, which could be measured from graph. EcoRI cut ‘s fragment was like straight elastic. It encounters more opposition migrating through a gel because it is spread out and will be in direct contact with more of the gel matrix. Uncut plasmid was tightly coiled, like a balled up elastic. While the molecule may be the same size, the coiling compresses it, leting it to meet less opposition when migrating through the gel. This indicated the minimal base brace of the plasmid.
If a intercrossed recombinant plasmid was constructed from pBR325 by the interpolation of a fragment of Deoxyribonucleic acid at the BamHI limitation site, foremost the entire size of the plasmid got bigger. To find the size of the inserted fragment, EcoRI and PstI were used to cut the plasmid, and consequence was compared with original plasmid ‘s fragment to acquire the size of the inserted fragment.
There was no extra set seeable behind the chief untrimmed plasmid set in lane 7. If there ware, they must be the chromosomal DNA sets, the absent of these in lane 2, 3, 4 and 5 was because they were excessively little, might non be seen, or ran off the gel.
The fluorescent sets of the DNA limitation fragments in lane 2, 3, 4, and 5 were several – crease brighter than the untrimmed plasmid set in lane 7. It was because that the DNA fragment are free at both terminals and can ‘uncoil ‘ and pick up every bit much EtBr as it fits, whereas the untrimmed plasmid could non uncoil more than a certain sum without the phosphate concatenation breakage: for every integrated molecule of EtBr it had an country of local ‘under-coiling ‘ that had to be compensated by another country of ‘over-coiling ‘ , this country would non incorportate any EtBr. So the figure of EtBr molecules it could pick up was limited because of sterical considerations. Therefore the DNA fragment had less sterical limitations, stained more than the untrimmed plasmid.