Copyright 2003

Introduction

The goal of this lab was to take the DNA that we had isolated and been working with last week and eventually obtain a sequence that we could cross reference with a library and collect information on the type of bacteria we had in our samples. In my case, sample 1 and 5.

	DNA Sequencing Methods
Reaction Set-Up Methods: In order to get different length fragments of DNA that stop transcribing at certain bases, we put our samples into four tubes, each of which had a different ddNTP. Since we know what base pair it will stop at, all we need to do now is figure out how long the fragment is and we can figure out the sequence.
Gel Pouring Methods: Before we pour the gel. The glass must be meticulously cleaned so that the machine can read it properly. Then, the two plates of glass are assembled using the brackets and the gel is poured in between them.

DNA Sequencing Results

Sample 1

aKtGAATTGTAATATGAGTcAcTATAGGGcGAATTGGGCccTcTAGATGcATGaTtGAGc

GGccGgcAGTGTGATGGATATtTGcAGAATTTGgCCTTGTGGGGGAAGGGTGAGTAAcAc

GTGGGAACCCGCCCCCCGGTGGGGGATAAcGGGACGaAAGTTGCGGTAATcCCGgATACG

TcCTTcGGGGGAAAGcGcAGTGcGGcCGGGGGAGGGGCCTgCGGcCATcAGGTcGTTGGT

GGGGTAAGGGGCTAcCAAGGCGATGACGGGTAGcTGGTCTGGGAGGATGACCAGcCAGAC

TGGGACTGAGAcAaGGcCCAGACTTCTACGGGAGGgAGgAGcAAGGAATTTTcGGgAATG

GGcGcAAGCTGACCGAGcAACGgCGgGTGcGGGATGACGGcCTTcGGGTTGTAAAcCGcT

TTTcGGGGGGACGACCCTGACGGTAaCCCCGGAACAAGCCCCGGCTAACCCTGTGcCAGC

AGCCGCGGTAAGACAGAGGGGGCGAGCGTTGTcCGGAGTcAcTGGGGcGTAAAGcGcGcG

cAGGcGGCAACCTAAGTGTcGTGTGAAAGcCCCcGGCTtAACCGGGGGAGGGCATGGgAA

AcTGGGTcGcTcGAGCTGcGGaGAGGGCCCTcGAATT

Sample 5

gTGAATTGTAATAtGAcTgAcTATAGGGcGAATTGGGctTtTAGATGTATGATTGAGcGG

ccGATAGTGTGATGGATATTTGGAGAATTTGGcCTTGTGGGGGAAGGGTGAGTAAAAAGT

GGGAAccCGCCCCCCGGTGGGGGATAAAGGGAaGaAAGTTGcGGTAATTtCGcATAAGTt

cTTTGGGGGAAAGGGGAGTGGGGGtGGGGGAGGGGGGTGcGGGGATTAGGTTGTTGGTGG

GGTAAGGGGGTAatAAGGGGATGAcGGGTAGGTGGTTTGGGAGGATGAACAGgCAGAaTg

GGAaTgAGAAAAGGGgCAGAATTTTAAGGGAGGGAGGAGgAAGGAATTTTTGGGAATGGG

GGGAAGGTGAAcGAGGAAAGgcGGGTGGGGGATGAAGGgGTTTGGGTTGTAAAAAGCTTT

TTGGGGGGAAGAcccTGAAGGTAAAaccGGAAAAAGGcctGGATAAATTTGTGGGAGcAG

GgGGGGTAAGAcAGaGGGGGGGcGGGTTGTTtGGaGTtAATGGGGGTAAAGGGgGGGGAG

GGGGGAAAaTAAGTGTTGTGTGaAAGGccCcGGcTTAAccGGGGGgGGGcATGGGAAAAT

DNA Sequencing Results

Sample 1
Accession 1	uncultured Chloroflexaceae bacterium	USA: Yellowstone	Boomer,S.M., Lodge,D.P., Dutton,B.E. and Pierson,B.
Accession 2	uncultured Chloroflexaceae bacterium	USA: Yellowstone	Boomer,S.M., Lodge,D.P., Dutton,B.E. and Pierson,B.
Accession 3	uncultured Chloroflexaceae bacterium	USA: Yellowstone	Boomer,S.M., Lodge,D.P., Dutton,B.E. and Pierson,B.
Accession 4	uncultured Chloroflexaceae bacterium	USA: Yellowstone	Boomer,S.M., Lodge,D.P., Dutton,B.E. and Pierson,B.

Sample 5
Accession 1	uncultured Chloroflexaceae bacterium	USA: Yellowstone	Boomer,S.M., Lodge,D.P., Dutton,B.E. and Pierson,B.
Accession 2	Cloning vector pPGKneo-II	artificial sequences	Graubert,T.A., Lu,Z.H. and Ley,T.J.
Accession 3	Cloning vector pPGKneo-I	artificial sequences	Graubert,T.A., Lu,Z.H. and Ley,T.J
Accession 4	Bacteria; environmental samples	swine intestine	Leser,T.D., Amenuvor,J.Z., Jensen,T.K., Lindecrona,R.H., Boye,M.

Discussion

The purpose of this lab was to figure out what the sequence was of our DNA. We did this by loading our ddNTP samples into a DNA sequencer, which was like loading a big gel, and the sequencer took pictures of the DNA fragments as it went past the scanner running off the gel. All of the information was stored onto a harddrive, and from there, we took the picture and used a software program to figure out the sequence. The program allowed us to view what it considered to be the proper base for each position and the readings that it found, and sometimes they came up as ambiguities. So we corrected them based on our own knowledge to try and come up with a more complete sequence. This proved to be pretty much useless for me, I was missing a lane from each of may samples so I did not get any of the C’s. I don’t know why this happened, and I am not sure if it was my fault, I would think if I did something wrong that my lanes would be empty or messed up, but they were not even there at all, like they were not even loaded, and I think that the likelihood of me forgetting to load the C group for both samples is pretty slim, so don’t know if it was a computer glich or what.