Tag Archives: Dungan isolates

Sequencing – Dungan Isolates, Lake Trout HRM and Emma DD cloning

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.

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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.

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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.

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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!

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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.

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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.

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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.

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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).

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