Copyright 2003

Introduction

The goal of these two labs was to take Red layer microbial mats, and perform PCR to amplify the 16s gene off all species present, and then to do a DGGE on the product of the PCR in order to separate out different species among the population of the microbial mat. Here is a picture of me drying the glass plates used in DGGE.

PCR

Results: I loaded the lanes labeled 1 through 4 on this gel. You can see a slight band in lane 1, which was supposed to be my negative control, there is nothing in lane 2, my 1x sample, lane 3 and 4 both have good bands, which were my 10x and 100x dilutions respectively. This means that I did get PCR amplification on these.

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DGGE

Methods: Here is a picture of our fully assembled DGGE gel box. Perfectly clean and sealed, isn’t it pretty.

Methods: Pouring the gel turned out to be pretty easy, despite the nature of the apparatus in the picture, which was looking pretty shaky to the untrained eye, but since we were under the supervision of trained professionals, I knew we were all safe and nothing would come crashing down on any of the students.

Results: Thanks to delicate handling, if you look closely at the mangled gel, you can see several distinct bands in my lanes, representing several different species of bacteria present in the microbial mat. Lane 8 was negative control, lane 9 was one to one, lane ten was 10x dilution and lane 11 was 100x dilution. Lanes 10 and 11 you can see 5 distinct bands, representing 5 different species, lane 9 has no bands, and lane 8 has one, representing a contaminated control. All of which is complementary to what I got form PCR results.

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Discussion

In this lab we used DGGE which is a technique to separate DNA strands based on their sequence composition rather than just size, as in PCR. Over 3 weeks, we prepared samples with PCR and did agrose gels as well as DGGE on them, and by comparing the two we find out what information can be obtained by doing a DGGE rather than just an agrose gel. The agrose gel in my experiment showed me that PCR was successful in the 10x and 100n dilutions, but was not in the 1x. it also showed me that I had replication taking place in by blank sample which must have been a result in contamination. The DGGE gel shows similar results, however the bands in the DGGE are distinct do to the different sequences of species of bacteria, and they formed 4 distinct bands, representing 4 different species of bacteria that had been amplified during PCR and present in the original sample of mat population. Realistically there are many more than that present in the population, which is why this technique should be done many times to get a good idea of the population, even then, some species are just in too few of numbers to ever really get a result for them. After DGGE the samples can be taken out of the gel and used again for sequence analysis, which seems like a pretty neat idea.