Submitted 1.5 plates for Sanger sequencing. Dungan isolates prepared by me, Lake Trout HRM prepared by Rony and Emma’s differential display cloning samples prepared by her. All primers were prepped by me. See the sequencing log for samples and plate layout.
Tag Archives: Dungan isolates
PCR – “Unknown” Dungans/Lyons
This is a repeat of yesterday’s set up with LABY primers, but with an annealing temp of 53C in hopes of improving the number of amplicons generated from additional samples. See yesterday’s PCR run for info on samples.
Results: Samples were loaded 1-29 and three negative controls from left to right, top to bottom.
The lower annealing temperature clearly resulted in more products. The ~500bp band was cut from each lane and stored @ -20C. All bands will be purified using Millipore spin columns and then sent for sequencing.
PCR – “Unkown” Dungans/Lyons
This was done on the numbered tubes using the LABY A/Y primers for eventual sequencing. Turns out many of the tubes have some info (other than just a number) on their sides which might provide more information regarding which isolate they actually are. PCR set up is here. Annealing temp 55C.
| Tube-# | Side Label |
| 1 | VA1423-1 |
| 2 | VA1423 2CB |
| 3 | VA1423-3 |
| 4 | VA-1423 4 |
| 5 | VA1423-6 6 |
| 6 | VA1423-10 |
| 7 | VA1423-12 |
| 8 | VA1423-15 |
| 9 | VA1423-26 |
| 10 | VA1423-28 |
| 11 | VA1423-290 2003 Isolate |
| 12 | VA1423 29 2004 Isolate |
| 13 | VA-1423-33 |
| 14 | VA1423-37 |
| 15 | XMAC13T |
| 16 | X-MAC-19T |
| 17 | XMAC 20T |
| 18 | X-MAD 10T |
| 19 | X-MAD-14T |
| 20 | X-MAD-18T |
| 21 | XMAE 11T |
| 22 | XMAE 13T |
| 23 | BC05CA8T |
| 24 | BC05CA 15T |
| 25 | BC05CA 18T |
| 26 | BC05CA 20T |
| 27 | 98 MFS 61A |
| 28 | CRT W 1HE/H11 |
| 29 | CRSH 5B3 |
Results: Samples were loaded 1-29 and three negative controls from left to right, top to bottom.
There are four prominent bands from Tubes 23, 27, 28, 29. These four bands were excised and purified with Millipore spin columns according to protocol. They will be sent for sequencing. There are faint bands visible from Tubes 9 & 11. Due to the faintness, they were not excised as there may not be enough product for sequencing. The remainder of the samples failed to produce any amplicons.
Sequencing – Dungan isolates
Submitted two plates for sequencing. Each sample two times from each direction.
PCR – Two new Dungan isolates
Repeat of yesterday’s PCR, but with AmpliTaq, less gDNA and 50uL rxn volume. PCR set up is here.
Gel loaded and run by Steven. Not entirely sure of the loading order. However, it doesn’t really matter…
Results: Still absolutely nothing.
UPDATE: Noticed on 20090713 that the reactions didn’t have dNTPs. Probably explains why it didn’t work!
PCR – Two new Dungan isolates from earlier today
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.
gDNA Isolation – Two new Dungan isolates
Isolated gDNA from the following two samples:
MIE-14y
VNTc-1.2-C1/G10
Samples were spun @ 16,000g @ RT for 2mins. No visible pellets in either sample. EtOH was removed. “Pellets” were washed in 1x PBS (pH=7.6) two times and then the Qiagen DNEasy Kit protocol was followed. Samples were incubated @ 55C with Proteinase K for ~2hrs.
Results: Both samples show really, really low quantities of gDNA.
PCR – Old Dungan isolates #1-35 w/EukA/B primers
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:
PCR – Dungan isolates from 20090402 with Euk primers
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.
PCR – New Dungan isolates
Repeat of PCR from 20090403, but using AmpliTaq and 50C annealing temp. PCR set up is here.
Lane 1 – Hyperladder
Lane 2 – 19t
Lane 3 – 100bp ladder
Lane 4 – 13t
Lane 6 – 17t
Lane 8 – 1.5t
Lane 9 – Hyperladder
Lane 10 – 1.2t
Lane 12 – 11t
Lane 13 – 100bp ladder
Lane 14 – H5
Lane 16 – 12t
Lane 17 – 100bp Ladder
Lane 18 – H2O
Lane 19 – H2O
Lane 20 – Hyperladder
Results: Nothing amplified! Possibly due to age of polymerase (?); over a year old. Will wait to repeat for new primers to arrive (EukA/B).