November 30 2018 DNA Extraction and Nanodrop – Soil C

Rationale: The purpose of this lab is to complete DNA extraction and quantify the DNA with the Nanodrop.

Description of Procedures:

The titer of the lysate was determined to be 2.0 x 10^10.
0.5 ml of sterile water was added to the DNA pellet to re-suspend it.
2 ml of 37 C Clean Up Resin was added to the pellet and swirled.
Resin pellet was transferred to 2 microcentrifuge tubes and spun for 3 minutes at 12,500 xg.
Supernatant pulled off with a bulb pipette and 1 ml of 80% isopropanol was added to each tube. The tube was spun at 12,500 xg for 3 minutes.
Step 4 repeated
Supernatant pulled off again and 1 ml of 80% isopropanol added to each tube. Liquid transferred to a two column syringe and allowed the vacuum to pull the liquid through the column to separate the DNA.
Column added to a clean microcentrifuge tube and spun at 12,000 xg for 5 minutes.
Transferred to a clean tube and 80 ul of 80 C elution buffer added to the tube. It was then spun in the centrifuge at 12,000 xg for 1 minute.
DNA was quantified using the Nanodrop.

Observations/Results:

DNA- 808.978 ng/ul
A260/A280- 2.049
A260/A230- 0.193

Interpretations:
The procedure was completed. The DNA was extracted and quantified. The A260/A230 value was low, meaning there could be too much guanidinium thiocyanate left in the DNA. The next step would be to perform PCR and run a gel, but there is not enough time left in the lab.

Extracted DNA and Spot Titer Test:

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December 1

Redoing of Nanodrop Results and Redoing PCR (11/30/18)

Rationale:

Gel electrophoresis was rescheduled for Monday. PCR will be done with the correct concentration of exacted DNA. Since the flash drive carrying the results from the Nanodrop was misplaced, it was decided to redo the Nanodrop readings. Also, more gels were made.

Nanodrop Results:

The results differed greatly from the previous ones. Instead of having a DNA concentration of 888.1 ng/µL, the following values were found.

Nanodrop Results from 11/30

Trial	Nucleic Acid (ng/µL)	A260/A280	A260/A230	A260	A280	Baseline Absorbance
1	1164.323	1.976	1.77	23.286	11.787	0.647
2	1077.553	1.993	1.725	21.551	10.811	-0.274
3	1121.914	1.979	1.751	22.438	11.338	0.836

Three trials were performed to confirm that the DNA concentration was much higher than previously recorded. All three trials had the same nucleic acid factor of 50 and the baseline correction of 340 nm. The final trial readings were recorded as Ferranti’s corrected Nanodrop results and were used in PCR calculations.

Procedure:

Combined and vortexed 1 µL of exacted DNA (Ferranti) with 9 µL of sterile water to create a Ferranti 10^-1
Combined and vortexed 1 µL of Ferranti 10^-1dilution with 9 µL of sterile water to create a Ferranti 10^-2
Once an aseptic zone was established, 12.5 µL of 1X MM, 2.2 µL of Ferranti 10^-2dilution, and 6.5 µL of ddH₂O were combined three times into three different microcentrifuge tubes.
4 µL of primer 1, primer 2, and primer 3 were placed into their correlating microcentrifuge tubes.
Placed microcentrifuge tubes in thermo-cycler and activated program STU.
40 mL of 1X TAE and 0.8 g of powdered agarose were combined and swirled together in an Erlenmeyer flask.
Heated the Erlenmeyer flask until the mixture was boiling then mixed solution until the bubbles disappeared. Repeated until the solution was consistent.
Allowed the solution to cool until it was cool enough to touch.
Added 2.0 µL of EtBr to the flask to achieve a concentration of 0.5 µg/µL.
Poured mixture into gel apparatus and placed comb.
Once the gel solidified, the comb was removed, and TAE buffer was poured over the gel to keep it from drying out.

Observations:

The thermocycler used the program STU which started with 5 minutes of initial denaturation at 98.0ºC. Then, 35 cycles of 30 seconds at 98.0ºC followed by 30 seconds at 55.0ºC followed by 45 seconds at 72.0ºC occurred. After the cycles, a final extension happened at 72.0ºC for 5 minutes.
A positive control was also made to confirm that the PCR mixture was not contaminated. Anita’s, a phage with a high titer of 2.010⁹pfu/mL, DNA was used.

PCR Calculations:

where

C₁is the extracted DNA concentration (1,121.91 ng/µL)

V₁is the volume needed to add into PCR mixture (needs to be ~ 2 µL)

C₂is the DNA concentration wanted (25 ng/µL)

V₂is the dilution factor

Steps 6-11 were repeated 4 times to create a total of 4 2% agarose gels.

Next Steps:

Gel electrophoresis will be run with the microcentrifuge tubes prepared from Wednesday (11/28) and the microcentrifuge tubes prepared today so both diluted and non-diluted samples can be compared to each other. Also, these results will identify Ferranti’s cluster.