Geochemistry & Ecology of Red Mat Systems (GERMS) Undergraduate Summer Research Program
Week Five DNA Sequence Analysis Jana K. Brooks

Sequencing Reaction Set – Up

Procedure:

Preparation of Strip Tubes For each of the 5 purified DNA clone templates a strip tube containing 4 tubes was prepared: Filling Left to Right: 2.0ul of ddTTP plus dNTPs 2.0ul of ddGTP plus dNTPs 2.0ul of ddATP plus dNTPs 2.0ul of ddCTP plus dNTPs
Mixing Cocktail with Clones 7.3ul of clone samples (Green tube) were combined with 9.7ul of a cocktail mix that contains buffer, primer and Taq polymerase. The resultant mixture is seen in the Pink tubes.
Loading Strip Tubes into PCR 4.0ul of the newly formed cocktail/clone mixture was added to the strip tubes containing the stop nucleotides. Because of the presence of different stop nucleotides in each tube, new tips were used for each addition of cocktail/clone mixture. Strip tubes were then placed in the PCR.

Preparing Gels: Jana, Jen, and Liz

Procedure:

DNA Sequencing analysis machine
Glass plates draining Be certain the plates (especially inner side) are clean, or residue will interfere with laser reading.
Preparing APS Catalyst .5 ml ddH2O is mixed with .05g APS and saved for addition to gel to influence polymerization.
Preparing Acrylamide Solution Measuring Urea 17.0 grams to be used to make the acrylamide solution for gel.
Preparing Acrylamide Solution Boiling Once the Urea is measured into beaker, it is barely covered in ddH2O. The mixture is boiled with stir bar until all Urea is dissolved.
Preparing Acrylamide Solution Dissolved solution is poured into a 50 ml graduated cylinder.
Preparing Acrylamide Solution To the Urea solution we added : 4 ml of 10X TBE 4.4 ml Long Ranger acrylamide solution Then the volume was brought up to 40 ml using ddH2O
Assembling Gel Apparatus Facing inner-side up, the surface of both glass plates was wiped down with 95% ethanol and Kimwipes®.
Assembling Gel Apparatus To facilitate the adhering of the spacers, small dots of ddH2O were positioned on the spacer prior to placing it along the side border of the back glass plate.
Assembling Gel Apparatus The front plate was positioned and clamped in place (snug but not too tight).
Assembling Gel Apparatus Using the holder, the plate apparatus was propped up at an angle to assist the gel pouring.
Pouring the Gel Catalysts were added: 270 µl APS and 27 µl TEMED Gel was quickly injected: Using a 60 ml syringe
Pouring the Gel “Tapping” action helps to move the gel frontage line move uniformly down the plates.
Gel Set-Up Once the gel front reached the edge, the gel was lowered to a horizontal position, and the comb was inserted upside down (flat side in).
Gel Set-Up Comb is secured with clamp and left to polymerize for 20 minutes
Gel Set-Up The gel has polymerized, and it is now time to get the wells ready.
Gel Set-Up Before the comb can be removed, ddH2O is used to clean off excess polymerized gel bits.
Gel Set-Up Cleaning the large well with TBA in a syringe.
Gel Set-Up The final step in cleaning the well is to run a fine piece of metal along the well without puncturing it to remove any bits of debris.
Lane Formation Finally the lanes are formed by turning around the comb and inserting it with the teeth pointing down.
Assembling Buffer Chamber Adding gasket to upper chamber
Assembling Buffer Chamber Placing Gel into sequencer
Assembling Buffer Chamber Filling lower chamber with 1X TBE
Assembling Buffer Chamber Connecting upper electrode
Assembling Buffer Chamber Connecting lower electrode
Assembling Buffer Chamber Upper connection complete
Computer Set-up Pre-load

Reaction Finish and Load:

Reaction Finish Magenta dye has been added to the Stop strip tubes
Reaction Finish Strip tubes are placed in the PCR machine so the DNA denatures
Loading Gel A fan-tip was used to load 1.5-2.0 µl of each reaction into a well. Detailed records were kept in lab books to delineate samples with their corresponding wells.
Gel Results Using e- Seq, each raw sequence was edited for ambiguities and then saved as a text document to import into BLAST

Reading, Editing and BLASTing Sequences

SEQUENCE INFORMATION

Clone Number Analyzed: HG 11

Edited Sequence:

TOP FIVE DIFFERENT BLAST HITS

Top Accession #	Organism Name	Source
AF239695	Gemmata-like strain	Soil, Freshwater
X81957	Gemmata obscuriglobus	Soil
AF121889	Bacterium 2BP-58	Community 2-bromophenol dehalogenating consortium
X62912	Pirellula marina	PCB-polluted soil
AJ292685	Uncultured eubacterium WD 283

Clone Number Analyzed: HG 14

Edited Sequence:

TOP FIVE DIFFERENT BLAST HITS

Be sure to use different hits (uncultured = same; go down until you find specific species, including cultured isolates)

For source, I am looking for where it came from (e.g. termite gut); if none is listed, read the title reference and abstract (if published) to glean this information.

Top Accession #	Organism Name	Source
AY647897	Hymenobacter sp. 29F	Biological soil crusts from the Sonoran Desert
AJ318187	Uncultured Bacteriodetes bacterium	Industrial biofilter
AF275712	Unidentified Hailaer soda lake bacterium Z3	Soda Lake
AY211113	Bacillus sp. ‘Mail 19’	Aerosolized bacteria and fungi from desert dust events in Mali, West Africa
AY423898	Flectobacillus sp. Td	Pink-pigmented bacteria isolated from paper mill slime

Clone Number Analyzed: HG 16

Edited Sequence:

TOP FIVE DIFFERENT BLAST HITS

Be sure to use different hits (uncultured = same; go down until you find specific species, including cultured isolates)

For source, I am looking for where it came from (e.g. termite gut); if none is listed, read the title reference and abstract (if published) to glean this information.

Top Accession #	Organism Name	Source
AJ488505	Muricoccus roseus	Indoor building materials of a children’s day care center
AJ292598	Uncultured eubacterium WD248	Biphenyl-polluted soil
AY150045	Roseomonas gilardii	Isolated from blood
AF465832	Rubritepida floccularis	Thermophilic source
AJ633644	Rhodovarius lipocyclicus	Alpha-1 subclass of Proteobacteria

Clone Number Analyzed: HG 18

Edited Sequence:

TOP FIVE DIFFERENT BLAST HITS

Top Accession #	Organism Name	Source
AP005376	Thermosynechococcus elongates BP-1	Thermo source
D83715	Synechococcus elongatus	Not available
AJ007374	Cyanobacterium WH7B	Great Sippeuissett Salt Marsh, MA, USA
X84808	Leptolyngbya foveolarum	Nonaxenic cultures
AY172830	Synechococcus sp. RS9920	Stratified water column in the Red Sea

Clone Number Analyzed: HG 19

Edited Sequence:

TOP FIVE DIFFERENT BLAST HITS

Top Accession #	Organism Name	Source
AF112998	Thiocapsa roseopersicina	Aerobic turnover of dimethyl sulfide
AJ292589	Uncultured eubacterium WD292	Polychlorinated biphenyl-polluted soil
AJ431219	proteobacterium BHI60-11	East Pacific rise at 13ºN
Y12301