Week Four

Geochemistry & Ecology of Red Mat Systems (GERMS)

Undergraduate Summer Research Program

Week Four – Practice

Genomic DNA Isolation and PCR

Genomic Isolation Day One:

Procedures: The object is to obtain high quality genomic DNA from the mat samples. The reaction works with no more than 0.1 g of mat sample. We kept the samples on ice. We lysed the cells, and extracted the DNA by beating the mat sample in a vial with Zirconium beads, 800 uL of Phenol/ Chloroform, 66 uL of 10% SDS, and topped of with GTE until no air space remains in the vial. After beating twice, we carefully removed the top aqueous layer above the red colored cell debris, shown in the bottom picture.

WEIGHING OUT 0.1G OF MAT

0.1G RED LAYER: FAIRY

LEFT: GREEN FAIRY

RIGHT: RED FAIRY

Results:

A small amount of precipitate formed after inverting the tube 40 times, shown in picture to the left. More appeared after freezing overnight.

FAIRY RED LAYER GENOMIC DNA ISOLATION

PCR:

Procedures: This first gel is to help us determine how much to dilute our samples by for PCR. We washed the DNA precipitate in 70% ethanol, inverted it and allowed it to dry. Then resuspened in 100 uL of 1X TE buffer. Then we made a separate 1/10X dilution by taking 5uL of the 1X, and adding it to 45 uL of dH20. WE set-up 6 reaction vessels each containing 25 uL of mastermix, 1 uL each of appropriate forward and reverse primers(16S or DGGE), 1 uL of DNA (any of three concentrations- 1X, 1/10X, 0), and 22 uL of dH2O. We stained the reaction contents on paraffin so as to not contaminate the rest of the product.

STAINING PCR PRODUCT ON PARAFFIN

PCR AGAROSE GEL

Gel Results:

1 2 3 4 5 6 7 8 9 10 11 12

Monica top section

Lane contents

1: 1x 16 S positive

3: 1x DGGE negative

5: 1/10 X 16S negative

7: 1/10X DGGE positive

9: NEG 16S negative

10:

11: NEG DGGE negative

12: MAKRER positive

DGGE:

Procedures: Wash plates twice, rinsing with distilled water. Spray with 95% ethanol, and dry with chem wipes. Fit with gasket and spacers on the back plate, and clamp together. A gradient maker (shown at bottom) was used to create a uniform gradient of high at the bottom, low at the top. Dcode dye was added to the High concentration side of the gradient maker so that the gradient could be visually seen. DGGE gel separates DNA based on GC content, which is relative to its melting point. Different members in a population will have different melting points. Thus, multiple bands indicate different members in the population.

CLEANING THE GLASS PLATES FOR DGGE ACRYLAMIDE GEL.

LOADING THE ARCYLAMIDE GEL

GRADIENT MAKER.

Gel Results:

16,15,14,13,12,11,10,9, 8, 7, 6, 5, 4, 3, 2, 1

LANES

1: X

2: X

3: X

4: R19- Gram negative Proteobacteria

5: H43- Gram positive Bacillius

6: C- Green-GNS

7: H- Red-GNS

8: JEN

9: MONICA- Fairy Red

10: MARIA-Fairy Green

11: H31- Gram negative Gamma Proteobacteria

12: H39- Gram negative Alpha Proteobacteria

13: S10- Cyanobacteria

Discussion: Lane 9 which is red layer from Fairy hot spring matches only with the band in lane 7 indicating red-GNS is present.

Plasmid Isolation (MiniPrep):

Procedures: Full length 16S PCR product was mixed with TOPO-TA and grown with Ecoli in media with ampicillin. The Ecoli has the ability to transform naked DNA into a vector
Gel Results: Monica’s		1 2 3 4 5 6 7 8 9 10 11 12
LANES :	CONTENTS:
1	Marker
2	blank
3	FR 1- vector only
4	FR 2- vector only
5	FR 3- vector only
6	FR 4- vector only
7	FR 5- vector only
8	FR 6- vector only
9	FR 7- vector only
10	FR 8- vector only
11	FR 9- vector only
12	FR 10- vector only	MINI PREP GEL

Discussion: No Ecoli picked up vectors with inserts from bacteria from the Fairy red layer, so that DNA can not be sequenced.