Aim of the experiment The aim of the series of the practical sessions was to transfer CIH-1, a fungal gene from pBK-CMV, a plasmid vector into pUC19, another plasmid vector. This was done by cutting the given fungal gene from pBK-CMV by use of Xba1 and EcoR1 that nick the plasmid from either side of the given fungal gene. The gene will then be ligated into pUC19. The ligated products eventually become transformed into E. coli and then there will be selection of bacteria colonies that will contain the recombinant pUC19 molecules. Confirmation Method In the first method involves restriction digestion of pBK-CMV and pUC19.
In this method, one was provided with RDMM (a restriction digest master mix) that contains, water, a restriction digest buffer and the restriction enzymes XbaI and EcoRI. Ligation method is then followed. After it, agarose electrophoresis of the pBK-CMV and pUC19 was done. This was then followed by the preparation of chemically competent E. coli. XLI blue. Agar plates were then prepared Results The following were the results got after doing agarose electrophoresis of pBK-CMV and pUC19. From the electrophoresis sheet, one could realize that some base pairs shifted to a higher distance from the base line while others short distance.
Therefore, the various distance between the top most part the base pairs reached from the base line recorded and values put in the table below. The results for the distance moved by the DNA size markers from the well included the following. DNA fragment size (base pairs) Distance moved from well (mm) 5000 10 3000 12 2000 15 1000 17 500 23 From the table above, it evident that DNA band with base pairs 5oo moved a distance of 23 mm, 1000 base pairs moved a distance of 17mm, 2000 base pairs moved a distance of 15mm, 3000 base pairs moved a distance of 12mm and 5000 base moved a distance of 10mm.
From these findings, one can deduce that the shorter the base pairs, the longer the distance they travelled and vice versa. Discussion From the above experiment, it is evident that DNA fragments with lower base pair migrated longer distances than those with more base pairs. Restriction endonucleases enzymes are basically enzymes that cut DNA at specific sites. The enzymes cut at four, six or eight base pair sequences of which are palindromic.
Sometimes restriction enzymes may cut in a middle of a recognition sequence, forming blunt ends, or commonly cut asymmetrically, resulting in sticky ends or stranded overhangs. DNA ligase come in hand to join the end pieces of the cut strands to create recombinant DNA molecule.
Please type your essay title, choose your document type, enter your email and we send you essay samples