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.