Geochemistry & Ecology of Red Mat Systems (GERMS)

Undergraduate Summer Research Program

 

Week Four – Practice: Genomic DNA Isolation and PCR

Imperial Runoff-Green

 

Elizabeth Jacobsmuhlen

 

 

Genomic Isolation Day One:

Procedures:

The picture to the right shows the green mat sample being weighed out at .1 grams.

The picture to the right is showing the removal of the top aqueous layer to a new 1.5 ΅L tube.  This layer then had an additional Phenol/Chloroform extraction performed.

Results:

Genomic Isolation Day One:

No DNA was visible to the naked eye.  The tube was placed in a -80oC freezer overnight.  The procedure will be continued tomorrow.

 

 

 

 

 

Genomic Isolation Day Two:

PCR Gel Results:

Procedures:

Samples were removed from the freezer from yesterday and prepared for loading onto the agarose gel, shown to the right.

Results:

From left to right:

Lane  1:  Blank

Lane  2:  1X 16S

Lane  3:  10X 16S

Lane  4:  Negative 16S Control

Lane  5:  Blank

Lane  6:  Molecular Weight Standard

Lane  7:  Blank

Lane  8:  1X DGGE

Lane  9:  10X DGGE

Lane 10: Negative DGGE Control

Discussion:

This gel was positive for 10X 16S as shown by the band in lane 3.  This gel was also positive for both 1X and 10X DGGE as shown in lane 8 and 9.  Lane 4 and 10 were negative as expected as they contained the negative controls for 16S and DGGE respectively.

 

 

 

 

 

 

 

 

 

 

DGGE Procedures:

To the right is the gradient gel apparatus for DGGE.  This gel will have a gradient change from 85% to 35%.  This allows for separation of DNA based on its genetic composition, particularly the concentration of G/C.  Much like a melting temperature the higher the %G/C, the higher the melting temperature.  The chemical composition of the gel will cause the DNA to break apart and the sample to stop in the position appropriate to its concentration of G/C.

The picture to the right shows the loading of sample or control into the DGGE gel.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DGGE Results:

Working from left to right:

Lane  1:  Blank

Lane  2:  Blank

Lane  3:  Blank

Lane  4:  R19 – Gram Negative    Proteobacteria (G-)

Lane  5:  H43 – Gram Pos. Bacillus (G+)

Lane  6:  C – Green - GNS

Lane  7:  H – Red - GNS

Lane  8:  Jana’s Sample

Lane  9:  Imperial Green 1X DGGE  (Elizabeth)

Lane 10: Imperial Green 10X DGGE  (Nate)

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

 

 

Plasmid Isolation (Mini Prep):

Procedures:

The picture to the right is the loading of sample into the Mini Prepraration or agarose gel set-up.

Results:

Insert only: 1500 base pairs

Vector only: 4000 base pairs

Insert + Vector = 5500 base pairs

Gel Description and Diagram:

MINI-PREP ANALYZED DATA

Lane (Nate):

1

2

3

4

5

6

7

8

9

10

11

Description:

I1

I2

I3

I4

I5

I6

I7

I8

I9

I10

M.W.S

Insert + or -

-

-

-

-

-

-

-

-

-

-(?)

n/a

 

Lane (Liz):

12

13

14

15

16

17

18

19

20

21

22

Description:

I11

I12

I13

I14

I15

I16

I17

I18

I19

I20

M.W.S

Insert + or -

-

-

-

-

-

+

+(?)

-(?)

+(?)

+(?)

n/a

 

 

Discussion:

The simple agarose gel, shown under the heading, PCR gel results, allows for the analysis of PCR product by a separation based on size.  This type of gel is a simple way to determine whether or not the primer used (16S or DGGE) was successful and that target template was amplified during PCR.  As can be seen above PCR was successful for 10X 16S (approximately 1500 base pairs) as shown by the band in lane 3; and for both 1X and 10X DGGE (approximately 300 base pairs) as shown in lane 8 and 9 respectively.  Lane 4 and 10 were negative as expected as they were both negative controls for 16S and DGGE respectively.  However, these base pair lengths are common in bacteria and do not indicate what specific organism is represented by the DNA template sample used, this type of gel merely shows whether or not PCR was successful.

The DGGE gel has a higher resolution than that of the simple agarose gel, it is able to indicate the diversity of the members present in the sample.  This technique is based on the composition of the DNA particularly the percent G/C, as discussed above.  As can be seen in the chart above, the data supports the presence of Red – GNS (Lane 7);  possibly Green – GNS (Lane 6); possibly G- Alpha Proteobacteria (Lane 12) although the comparison line in Lane 9 is faint; and possibly either Gram Positive Bacillus (Lane 5) or G- Gamma Proteobacteria (Lane 11) due to the fact that the lanes do not line up precisely.

The plasmid isolation/mini preparation gel is used to determine which clones had successfully incorporated target insert into the vector.  As can be seen with the gel and chart above only one, I16, can strongly support that both insert and vector are present in the clone sample ran on the gel.  Others as indicated by the chart were difficult to determine if insert was or was not present in the vector.  This gel helps in making the determination which colonies of plasmid should be used for DNA sequencing.