Geochemistry & Ecology of Red Mat Systems (GERMS) Undergraduate Summer Research Program
Week Four – Practice Genomic DNA Isolation and PCR Jana K. Brooks

Genomic Isolation Day One:

Hillside Green Mat Sample Amount of sample for Hillside Green was .0704grams
Jen weighing Hillside Red Mat Sample
Obtaining Phenol/Chloroform The phenol is in the bottom layer
BB tube, ready to beat The bead-beating functions to lyse open the cell wall and membrane, while effectively cleaving the bonds between membrane proteins and the DNA
BB tube on ice between beating intervals
Centrifuge, spinning BB tube for 10 minutes Functions to separate material based on weight
BB tube after The DNA is located in the aqueous layer on top. The middle layer is the organic layer The bottom contains the beads and cell membranes and proteins
Tube inverted This acts to ppt out the DNA
Results: See the DNA! The DNA is 90% pure in buffer

Genomic Isolation Day Two: Now that we have a pure sample of DNA, we must amplify the amount in order to have the amount we will need to process on different gels. This amplification is done using PCR.

1X TE Buffer Volume of DNA was brought up to 200µl with 1X TE buffer
FL16S Forward and Reverse Primers
DGGE Forward and Reverse Primers
Master Mix Contains TAQ DNA polymerase dNTPs
Nuclease-Free Water
Suspending Samples
Strip Tube 1) 1X 16S 2) 1X DGGE 3)1/10 X 16S 4)1/10 X DGGE 5) negative control 16S (uses nuclease-free water instead of template) 6) negative control DGGE (uses nuclease-free water instead of template)
PCR Machine Allows a segment of DNA between two defined priming sites (via “flanking primers”) to be amplified. By going through cycles of boiling and cooling with an ample supply of TAQ (high temperature DNA polymerase from Thermus Aquaticus), and dNTP monomers, the DNA strand is exponentially copied. Because of the specific primers used, the results will be the Bacterial DNA isolated for 16s and DGGE. The exponential copies will be used for the gels

Dye on parafilm 2µl of dye was mixed with 10µl of sample and then loaded into a well in the gel
Loading PCR Gel
Loading PCR Gel
Running PCR Gel
Results of PCR Gel Separates by size 16S=1500bp DGGE= 300bp Top row is mine: 1) 1X 16S 2) 1X DGGE 3)1/10 X 16S 4)1/10 X DGGE 5) negative control 16S (uses nuclease-free water instead of template) 6) negative control DGGE (uses nuclease-free water instead of template) The 16S band is easily seen in row 3, and as expected there were no bands in rows 5 or 6

Discussion:

Because the PCR gel separates by size, it was expected that the 16S bands (1500bp) would not go as far on the gel as the DGGE bands (300bp). With the results from this gel, the PCR products from strip tube #4 will be used in the DGGE gel.

DGGE Procedures:

This gel separates based upon GC content. The higher amount of GC base pairs in the DNA sample, the harder breaking that segment of DNA will be because they are held together by 3 hydrogen bonds, as opposed to AT base pair bonds that are held together by 2 hydrogen bonds. The top of the gel with a low concentration of melting chemical should stop segments that have low GC content, and the bottom that has a high concentration of melting chemicals in the gel will contain segments with high GC content. The number of bands is indicative of the number of individuals in the population.

Cleaning Glass Plates and Drying with EtOH
Attaching Gasket
Clamping System Together
Danny Lecturing
High and Low Stock Solutions Must be kept on ice
Gradient Maker By using the toggle switch in the middle, this tool will form a concentration gradient throughout the gel
Stream of Gel
One Team Pouring Gel
Gravity Gel- Pouring Station
Comb Placement This must be done relatively quickly one the form is filled, as it begins to quickly polymerize. Were oxygen to get in it would interfere with this process, so saran wrap and clamps are positioned at the top to prevent air from entering and to hold the 2 plates firmly together.
Getting Gels Ready to Load Before inserting into the gel holder, you must pull away the gasket at the lower end so that the gel can run correctly.
Submerging Gel in Buffer Tank The gel must be in the buffer tank before it can be loaded

DGGE Results and Gel:

Lane 1: Blank	Lane 9: Liz, IG 1X DGGE
Lane 2: Blank	Lane 10: Liz, IG 10X DGGE
Lane 3: Blank	Lane 11: H31
Lane 4: R19, Gram -	Lane 12: H39
Lane 5: H43,	Lane 13: S10
Lane 6: C	Lane 14: Blank
Lane 7: H	Lane 15: Blank
Lane 8: Jana, HG 1/10 x DGGE	Lane 16: Blank

Discussion:

Lane’s 4-7 and 11-13 are the controls. The controls act as markers. If our samples are matched with a control we determine that they are the same organisms. My lane, lane 8, has one band that lines up with the band in lane 7, which contains the control Red-GNS. There is some error, since the lines don’t match up perfectly.

Plasmid Isolation (MiniPrep):

Is there Plasmid Incorporated into the Vector? I was given 10 clones from Hillside Green (11-20) that were obtained from taking 16S products mixed with a gluing agent called TOPO-TA and E. coli. E. Coli’s plasmid has ampicillin resistance. This mixture was then grown on ampicillin media. I transferred 2ml of each clone into 10 tubes
DNA Pellet Drying
Loading Gel with Clones and a Marker

Mini-Prep Gel Description and Diagram The top block represents vector only, such as seen in Lane 11 Of the top row, only lane 9 has vector and plasmid Top Row Jen’s Hillside Red 1-10 11: Marker Bottom Row Jana’s Hillside Green 1-10 11: Marker 1) H11 2) H12 3) H13 4) H14 5) H15 6) H16 7) H17 8) H18 9) H19 10) H20

MINI-PREP ANALYZED DATA

Clone #	EcoRI Product(s) #, sizes	Insert?
HG11	Vector + insert	yes
HG12	Vector
HG13	Vector
HG14	Vector + insert	yes
HG15	Vector
HG16	Vector + insert	yes
HG17	Vector
HG18	Vector + insert	yes
HG19	Vector + insert	yes
HG20	Vector

Discussion: This gel easily shows which lane has the vector plus insert and which is solely vector. The bands that are only vector appear at 4Kb, while the ones that have vector and insert are larger, and appear at 5.5Kb. Lane 6 and 9 have Vector and insert, but because there are two bands, there are two different inserts.