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Extraction and analysis of genomic DNA

ABSTRACT

Samples of E.coli DNA, either digested or undigested with different restriction enzymes EcoR1 and Sau3AI, together with DNA extracted from worm, liver and de-shelled pea using SDS-lysis technique will be quantified. The quality of DNA present in the DNA samples will be estimated after gel electrophoresis by comparison of the migration of supercoiled (RF1), circular (RF2) and linear (RF3) plasmid molecules. Overall it was a successful experiments where the findings corresponded to the expected results.

INTRODUCTION

As little as 4,000 genes in their genome, this is the number of genes that small organisms such as bacteria could carry whereas large organisms where as humans can carry up to (encode) more or less 30,000 genes. These numbers are very important tools for the order, type and number of genes found in a particular organism the function and characteristics of particular organisms can be verified. Made up of DNA or RNA genomes length is determined by its length and is given as base pairs (bp) (Genetic Engineering Laboratory Manual, 2011).

Through techniques such as extraction of DNA and agarose gel electrophoresis molecular biologists can manipulate and study gene identification. Investigation of the differences in genome structure are attained through using digesting enzymes where the enzymes are used to generate series of DNA fragments whose number and length depends on the size and shape of the genome (Genetic Engineering Laboratory Manual, 2011). Thus the fragments then can be analysed by gel electrophoresis (the most widely used and useful method) to separate and purify fragments (Sadava et al., 2010) , the patterns obtained after electrophoresis can be used to determine whether the genome is single or double stranded and whether it is DNA or RNA.

RESULTS

DNA sample pUC18 undigested (i.e. lane 1 on fig.1) shows 1 band apparent linear form and determined as ~2650bp. DNA sample pUC18 EcoR1 digest (i.e. lane 2 on fig.1) shows 1 band apparent linear form (DNA corresponds to the size of the plasmid) and determined as ~2650bp. DNA sample Bacterial DNA undigested (lane 3 on fig.1) shows a smear of genomic DNA and 1 band apparent of catena ted circular plasmid DNA and determined as greater than ~ 10,000 bp. DNA sample Bacterial DNA Sau3AI digested (lane 4 on fig.1) shows a blank. Liver genomic DNA (lane 5 on fig.1) shows a smear (genomic DNA). Worm genomic DNA (lane 6 on fig.1) is blank together with Pea genomic DNA (lane 7 on fig.1) which is also blank. Last but not least lane 8 shows 1000bp ladder.

Figure 1: Agarose gel electrophoresis of chromosomal DNA. Lanes 1, pUC18 undigested; 2, pUC18 EcoR1 digest; 3, Bacterial DNA undigested; 4, Bacterial DNA Sau3AI digested; 5, Liver genomic DNA; 6, Worm genomic DNA; 7, Pea genomic DNA; 8, 1000bp ladder (see below fig.2).

{N.B. 1- 8 is from left to right}.

Figure 2: Agarose gel marker, 1000 base pair ladder.

DISCUSSION:

For lane 1 : DNA sample pUC18 undigested showed 1 band that was linear however in the prelab it was predicted that two lanes should be expected, one that is super coiled and one that is linear. Having just the one band it is clear that an experimental error must have occurred.

However for pUC18 digested with EcoR1 digest (lane 2) was what was expected, 1 band which is linear which corresponds to the size of the plasmid. For lane 3 (Bacterial DNA undigested) there was a very faint smear evident and 1 band. This is said to be genomic DNA smear and was expected in the experiment. As for lane 4 (Bacterial DNA Sau3AI digested) was blank, this is due to the fact the Sau3AI cuts a 4 bp sequence of GATC which is much more common than a 6bp sequence, thus there were more cuts which leads to many sized DNA fragments both small and large. This leads to further migration because they are much smaller fragments; this was expected in the experiment.

Lane 5 had a genomic DNA (smear) which was large this is due to the increase in molecular weight. So as the molecular weight increases of the genomic DNA so does the smear. The blank lanes for lanes 6 and 7 were not expected for they should have been very similar to lane 5 for these lanes where all genomic DNA from different sources. Having said this lane 5 (would have had large smear together with lane 6) and lane 7 slightly smaller smear. (Genomic DNA large fragments mean large MW and smear).

Last but not least lane 8 was what was expected for it was the 1000bp DNA ladder. Overall I was happy with the experiment and it is clearly evident that experimental error occurred especially for lane 6 and 7. It is also possible to state that gloves where not worn at all times whilst the experiment was conducted.

Refernces:

2011 Genetic engineering laboratory manual/ Philip O’Brien, Ravi Tiwari, James Haile & Mike Bunce

Sadava, et al., Life: The Science of Biology, Ninth Edition, Sinauer Associates

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