Deja Vu Birney's TRAIN Blog

       Last week in lab we had run our left fragment from   Deinococcus radiodurans,   and our tetracycline fragment from   E. coli.  On a heat gradient from 34 ° C to 54 ° C. This gradient led to our discovery of the ideal temperature for a fragment overlap, that being 54 ° C. With this, we decided to play around and do another gradient. This gradient was from 52 ° C to 70 ° C with a total of 8 reactions. We did have to make more left and tetracycline fragments as we were running low, so we had spent most of our time amplifying those samples and gel excising them. For our overlap reactions, we used the recipe below:   5µL 7µL 11µL 1µL Left Tet Master Mix PCR H 2 O   We ran these 8 samples through PCR in their gradient. We then amplified the samples using the recipe below:   5µL 10.75µL 2.25µL 2µL Overlap Sample Master Mix PCR H 2 O Primer Mix  (A & D)   We then run these 8 samples on a big 1.2% gel for about an hour. ...

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 H 2 O   With this ...