Tag Archives: gel extraction

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.

PCR – C.pugetti gDNA from 20090526

This is a repeat of yesterday’s PCR due to the presence of bands in the water-only samples. Will use reagents and universal 16s bacterial primers (27F & 1492R) provide by the Horner-Devine lab in hopes of: 1) getting this two work and, 2) figuring out the source of the contamination.

All rxns were prepared sterily and all instruments, racks, tubes, tips and water were UV-sterilized for ~45mins in the biological hood. Rxns were prepared in the biological hood. PCR setups are here. Anneal 60C. Cycling params same as yesterday.

Lane 1 – 100bp ladder

Lane 2 – DNA (HD Rxn 1)

Lane 2 – H2O (HD Rxn 1)

Lane 3 – H2O (HD Rxn 1)

Lane 4 – DNA (HD Rxn 2)

Lane 5 – H2O (HD Rxn 2)

Lane 6 – H2O (HD Rxn 2)

Lane 7 – DNA (SR Rxn)

Lane 8 – H2O (SR Rxn)

Lane 9 – H2O (SR Rxn)

Lane 10 – 100bp ladder

Results: Well, we got our band and NO contamination in any H2O lanes. The super-bright, 1500bp band will be excised and purified using Millipore spin columns and submitted for sequencing. However, this gel is interesting because the primers provided by Mike (used in HD Rxn 1 and SR Rxn) did not amplify anything…

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

Ran PCR with GoTaq on the new Dungan isolate gDNA from yesterday. PCR set up is here. 53C annealing temp.

Lane 1 – Hyperladder

Lane 2 – 17t

Lane 4 – H5

Lane 6 – 12t

Lane 7 – Hyperladder

Lane 8 – 1.5t

Lane 10 – 1.2t

Lane 12 – 11t

lane 13 – Hyperladder

Lane 14 – 13t

Lane 16 – 19t

Lane 18 – H2O

Lane 19 – Hyperladder

Lane 20 – H2O

Results: All the bands present are a bit larger than 400bp. However, the bottom band in the H5 sample is larger than any band present in any other samples. Additionally, the largest band in the H5 sample is between 800-1000bp. Bands were cut out from H5 (two bands: Top, Bottom), 1.5t, 1.2t, 13t. These will be purified and sequenced.

It’s also interesting to note that the bands present in this gel are found in the same samples as the last time this analysis was done. See Kevin’s Notebook, 20090212.