34°C to 54°C Gradient on Left and Tet Fragments

This week, we did a PCR overlap heat gradient ranging from 34°C to 54°C on our left fragment of deinococcus radiodurans and our tetracyline fragment from E. coli. When we ran this reaction on a gel, there was absolutely no DNA showing up. We assumed there was maybe a problem with the pipetting of the fragments into the tubes when they were going through PCR for amplification, and we decided that it would be best to start from the beginning again to ensure that there was no error. We then proceeded to redo the overlap as well as the amplification process at 34°C to 54°C. For these reactions, we made sure they were equal molar. We first had to dilute our samples down to left being 10ng/µL and tet being around 5ng/µL. We did this by getting our original samples and adding PCR water. With our goal of having a 50ng/µL total for left and a 35ng/µL for tet, we loaded the amount below.

 

5µL 7µL 11µL 1µL

Left Tet Master Mix PCR H2O

 

With this being our PCR reaction, we repeated this recipe 8 times with every sample within our 34°C to 54°C gradient. In our PCR machine, we placed them with reaction #1 being the highest temperature, and reaction #8 being our lowest temperature. We also included our “Mary” reaction (Left, Tet, and Right at 44°C) that had the recipe below. 

 

5µL 5µL 7µL 11µL

Left Right Tet Master Mix

 

 

Although our “Mary” sample had a volume higher than all our other reactions, we didnt worry about it too much as our confidence in this reaction working was low. We ran PCR overlap on the samples for 90 minutes. After the 90 minutes passed, we amplified it at 60°C for 90 minutes as well. 

 

5µL 10.75µL 2.25µL 2µL

Overlap Sample Master Mix PCR H2O Primer Mix  (A & D)

 

On Friday, we finally ran our samples on a large gel. This large gel was put in the order listed below.

 

 

|  L
|
|  #1 - 54°C
|  #2 - 52.3°C
|  #3 - 49.7°C

|  #4 - 46.3°C
|  #5 - 41.7°C
|  #6 - 37.7°C
|  #7 - 35.3°C
|  #8 - 34°C
|

| Mary - 44°C

 

When reviewing our gel, we could see a band at 1700bp (Left and Tet Fragment) at reactions #1-#8. The band at 54°C was our brightest band so we can assume that 54°C is the most ideal temperature for overlap of left and tet. Next week, we will be loading the rest of our Reaction #1 sample on a gel to get a better look at the 1700bp band and any ghost bands that we could vaguely see.

Gel w/ reactions #1-#8 and Mary Sample
(from L to R)