Sequencing

March 1, 2022

Resuspending Primers

1. Locate your primers in the package and ensure they are the primers you ordered.

2. Compare the nucleotide sequences and GC% on each primer label to the order invoice. The melting temperature will likely differ between the label and the order (this is not important).

3. Spin down the tubes using the centrifuge machine. SPIN ALL TUBES BEFORE OPENING! This is because dry pellet can often come dislodged during shipping and could be in the cap.

4. Create a 100uM master stock by adding MBG water into the tube. Take the amount of nanomoles (nmols) on the label and multiply it by 10. That number is how many microliters of MBG water you'll add to the tube.

Added 301 uL of MBG water to KCNJ5_seq_FOR7 primer.

Added 263 uL of MBG water to KCNJ5_seq_REV3 primer.

5. Label the top of each primer tube with its name and concentration (100 uM).

6. Wait 10 minutes to allow the master stock to rest at room temperature and then mix well (centrifuge) before creating the working solutions.

We then made 30uL of 5uM solutions for both sequencing primers. We added 1.5uL of 100uM stock to 28.5 uL MBG water.

Nano dropping

1. We nano dropped our Maxiprep products. Plasmid DNA Amounts:

   1. 1. Premix 1: 5140 ng/uL

      2. Premix 2: 3337 ng/uL

      3. Premix 3: 9507 ng/uL

2. Our plasmid is 6650 bps, so the desired template concentration is 66 ng/uL

Results

Our sequencing results indicated that our DNA was properly inserted. The areas highlighted in blue are the segments that were sequenced. Both the beginning and end sequences matched our plasmid map, indicating that the DNA was properly inserted. Premix 1 gave the best sequencing results, towards the end of premixes 2 and 3, there was increasing uncertainty. At the end of premix 3, the sequencing ligase seems to have added in an extra adenine at position 2928. This did not impact the sequencing of our DNA's insertion into the plasmid.