Geochemistry & Ecology of Red Mat Systems (GERMS)

Undergraduate Summer Research Program

 

Week Four – Practice

Genomic DNA Isolation and PCR

 

 

Genomic Isolation Day One:  Site, Hillside

 

Procedures:  Began by weighing out 0.1g of mat.  Prepared the Bead Beating tubes with the beads and the 800microLiters of Phenol/chloroform.  After adding mat sample, 66microLiters of SDS and topping off with GTE the bead-beating can begin.  The purpose of this process is to break the cell wall, plasma membrane, and separate the DNA from the proteins that hold it to the plasma membrane.

Weighing the sample

All reagents are mixed and ready to start bead beating

Results:  After several rounds of centrifuging and additional reagents the DNA can be seen coming out of the solution—it is white, globular, strings of clumps!

See the DNA!!

 

 

PCR:

 

Procedures:  This is a process of DNA amplification using a master mix containing Taq polymerase and dNTPs, primers and the DNA that was previously extracted from the bacteria samples.

Procedures:  It was separated into 6 tubes, two are negative controls and the other four contain specific primers that will narrow the amount of the DNA to be copied.

Results:  Because of the specific primers used the results will be just the Bacterial DNA that we want to study, 16s and DGGE.  Later these samples will be used to run our gels.

Gel Results:  The PCR gel to the right is telling us whether or not we had DNA amplification take place. 

 

The top row is Jana’s.

The bottom row is mine.

 

 

Lane 1

1X 16S

Negative

Lane 2

1X DGGE

Negative

Lane 3

1/10X 16S

Positive

Lane 4

1/10X DGGE

Positve

Lane 5

Negative control 16S

Negative

Lane 6

Negative control DGGE

Negative

Lane 7

Marker

Positve

Lanes 8-12 are all blank

 

Discussion: 

 

The results of the PCR gel are just showing that I either got a positive result or a negative.  The positive result means that I had DNA amplification and the negative result means that I had no DNA amplification.

 

 

 

 

 

 

 

 

 

 

 

 

 

DGGE Procedures:

 

Step 1:  Preparing the equipment, washing.  The glass plates have to be really clean so that the gel doesn’t get caught on anything while flowing in.

Step 2:  Setting up equipment, putting together all the parts. 

Step 3:  Preparing the High and Low concentration gradients for the gel and loading into apparatus

Step 4:  Let the gel flow

Step 5:  Putting the comb in the gel to make the wells while the gel polymerizes.  You don’t want any oxygen to get in, hence the saran wrap and clamps at the top, otherwise the gel won’t polymerize correctly and the wells won’t form correctly either.

Step 6:  After the gel has polymerized it can be put in the buffer solution and the samples can be loaded.

 

 

 

 

 

 

 

 

 

 

DGGE Results:

 

Lane 2 

Blank

Lane 3

Blank

Lane 4

R19  gram negative proteobacteria(G-)

Lane 5

H43  gram positive proteobacteria(G+)

Lane 6

C      Green-GNS

Lane 7

H      Red-GNS

Lane 8

Jennifer’s sample  (mine)

Lane 9

Monica’s sample

Lane 10

Maria’s sample

Lane 11

H31  G – gamma proteobacteria

Lane 12

H39 G – alpha proteobacteria

Lane 13

S10  Cyanobacteria

Lane 14

Blank

Lane 15

Blank

Lane 16

Blank

 

Discussion: 

 

Lane’s 4-7 and 11-13 are our controls.  The purpose of the controls is to act like markers so that our samples bands in the middle of the gel can be lined up with the controls to determine the different organisms in our samples.  My lane, lane 8, has one band that lines up with the band in lane 7, which contains the control Red-GNS.  Since the bands line up fairly closely, this data supports that lane 8 has the Red-GNS organism.     

 

 

 

Plasmid Isolation/ Mini Prep:

 

Procedures:  We received 10 tubes containing cloned material.  The purpose of this process is to extract the newly cloned DNA and run a gel on it to see whether or not the plasmid (previously added) has been incorporated into the vector.

Procedures:  To the right, our extracted DNA dries before we can resuspend, add the enzyme and incubate before running the gel.

Procedures:  To the right, Jana and I load our gel.

 

 

 

 

 

 

 

 

 

Results:  The top portion of the gel (labeled 1-11) is mine and the bottom is Jana’s.

 

The top block represents vector.

 

 Based on these results I had one clone, in lane 9, which had both the vector and plasmid.

Lane 1

 

Hillside Red 1

 

Lane 2

Hillside Red 2

Lane 3

Hillside Red 3

Lane 4

Hillside Red 4

Lane 5

Hillside Red 5

Lane 6

Hillside Red 6

Lane 7

Hillside Red 7

Lane 8

Hillside Red 8

Lane 9

Hillside Red 9

Lane 10

Hillside Red 10

Lane 11

Marker

 

 

MINI-PREP ANALYZED DATA

Clone #

EcoRI Product(s) #, sizes

Insert?

HR1

One

Negative

HR2

One

Negative

HR3

One

Negative

HR4

One

Negative

HR5

One

Negative

HR6

One

Negative

HR7

One

Negative

HR8

One

Negative

HR9

Two

Positive

HR10

One

Negative

 

Discussion: 

 

Week 4 was spent tirelessly working on DNA extraction, amplification, cloning and running gels.  For the PCR gel, we got what was expected for DNA amplification.  I had hoped that the DGGE gel would show a more diverse community, however, I only got one band that I could see and, therefore, only one organism (that was recognizable).  There could have been other organisms in the community that just didn’t show up on the gel.