PCR of MIE-14v just to make sure that we can’t get a product from this sample, despite NanoDrop readings suggesting that there’s no DNA. Used both LABY and Euk primer sets. PCR set up is here. Anneal temp 50C.
Lane 1 – 100bp Ladder
Lane 2 – Euk
Lane 3 – Euk H2O
Lane 4 – Euk H2O
Lane 5 – Euk H2O
Lane 6 – LABY
Lane 7 – LABY H2O
Lane 8 – LABY H2O
Lane 9 – LABY H2O
Lane 10 – 100bp Ladder
Results: Nothing, as expected. Need to devise a new method of isolating gDNA from these “problem” isolates.
Set up PCRs on:
MIE-14y
VNTc-1.2-C1/G10
Used Euk A/B and LABY A/Y primers. Anneal temp 50C. PCR set up is here.
NOTE: Due to the extremely low concentrations of gDNA from these two samples, I used a large amount of gDNA in the rxns. Check the PCR set up link for actual numbers.
Lane 1 – Hyperladder (5uL)
Lane 2 – VNTc-1.2-C1/G10 (Euk primers)
Lane 3 – MIE-14y (Euk primers)
Lane 4 – H2O (Euk primers)
Lane 5 – H2O (Euk primers)
Lane 6 – VNTc-1.2-C1/G10 (Laby primers)
Lane 7 – MIE-14y (Laby primers)
Lane 8 – H2O (Laby primers)
Lane 9 – H2O (Laby primers)
lane 10 – 100bp Ladder
Results: Nada. Probably because of low [gDNA], but could also be due to PCR inhibitors in the gDNA. Will retry using Amplitaq and less gDNA.
Steven had me re-PCR the old Dungan isolates with the new EukA/B primers. Anneal temp 50C. PCR set up here (bottom half of sheet) .
NOTE: Sample #30 was not in the rack of tubes that Steven gave to me.
Results:
Did PCR with new Euk primers designed by Steven. Should be one step higher taxonomically. PCR set up is here. Aneal temp 50C.
Lane 1 – Hyperladder
Lane 2 – 19t
Lane 4 – 13t
Lane 6 – 17t
Lane 7 – 100bp ladder
Lane 8 – 1.5t
Lane 10 – 1.2t
Lane 11 – Hyperladder
Lane 12 – 11t
Lane 14 – H5
Lane 15 – 100bp ladder
Lane 16 – 12t
Lane 18 – H2O
Lane 19 – H2O
Laen 20 – Hyperladder
Results: The new EukA/B primers worked wonderfully. The brightest band in each lane was excised and purified using Millipore DA spin columns. These will be stored and sequenced at a later date.
