Tag Archives: PCR

PCR – Sepia cDNA (from yesterday)

Set up PCR on recent Sepia cDNA samples using both S. officianalis_rhodopsin_F, R primers & Sep_op_F2, R2 primers. PCR set up is here. Looking back at my old paper (gasp!) notebook from March 2007, I noticed that each primer set required differing amounts of Mg2+. Rhodopsin primers require 3mM Mg2+ in the PCR rxn and the opsin primers require 2mM Mg2+ in the PCR rxn. Mg2+ was added as required and is shown on the PCR set up link above.

Results:

Gel Loading (from left to right):

Opsin Primers (lanes 2-10), Rhodopsin Primers (same loading order, lanes 12-20)

1 – 100bp ladder

2 – retina

3 – fin

4 – 4th arm

5 – dorsal mantle center

6 – dorsal mantle side

7 – ventral mantle center

8 – ventral mantle side

9 – H2O

10 – H2O

Opsin Primers

We see an intense band in the retina sample, as expected, since opsin is constitutively expressed in this tissue. We also see a band in the fin and both dorsal mantle samples.

Negative controls are blank.

Rhodopsin Primers

We see an intense band in the retina sample. We also see a band in the fin sample. We see two bands in the ventral mantle center tissue, possibly suggesting a rhodopsin isoform is also being expressed in this tissue.

Negative controls are blank.

Overall, these results are rather intriguing because they clearly demonstrate that both genes are being differentially expressed in non-eye tissue of Sepia officianalis.

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 – Dungan isolate (MIE-14v) gDNA from 20090708

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.

PCR – Bay/Sea Scallop DNA

An additional attempt to get the actin primers to work for use in screening samples for bay/sea scallop hybrids. The scallop_actin_fw primer was used in conjunction with the following:

bay_actin_Rv0 (Rxn 1)

bay_actin_Rv2 (Rxn 2)

sea_actin_Rv4 (Rxn 3)

sea_actin_Rv5 (Rxn 4)

PCR set up is here. Just used Bay or Sea scallop gDNA (chelexed). When/If get this working correctly, will start screening the hybrid samples. Anneal of 53C.

Lane 1 – 100bp Ladder

Lane 2 – Rxn 1: Bay

Lane 3 – Rxn 1: Sea

Lane 4 – Rxn 1: H2O

Lane 5 – Rxn 1: H2O

Lane 6 – Rxn 2: Bay

Lane 7 – Rxn 2: Sea

Lane 8 – Rxn 2: H2O

Lane 9 – Rxn 2: H2O

Lane 10 – Rxn 3: Bay

Lane 11 – Rxn 3: Sea

Lane 12 – Rxn 3: H2O

Lane 13 – Rxn 3: H2O

Lane 14 – Rxn 4: Bay

Lane 15 – Rxn 4: Sea

Lane 16 – Rxn 4: H2O

Lane 17 – Rxn 4: H2O

Lane 18 – 100bp Ladder

Results: Rxn 1 shows amplification with both Bay & Sea Scallop gDNA. The bands are close in size, but look like they would be more distinguishable if run on higher percentage gel and for a longer period of time to get better separation. However, there is contamination in one of the two water samples..

Rxn 2 shows amplification of only the Bay Scallop gDNA.

Rxn 3 shows amplification in both Bay & Sea Scallop gDNA and both bands are of the exact same size.

Rxn 4 shows no amplification in either set of gDNA.

Using the primers used in Rxn 1 will probably allows us to succesfully screen potential hybrids. Just need to remember to use high-percentage agarose gels and run samples for longer periods of time to get sufficient separation.

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 – C.pugetti gDNA from 20090513 & 20090526

Previous PCRs from 20090601 & 20090602 both showed contamination in the negative control. Suspect that the primer stocks were contaminated due to the usage of older, non-sterile TE for reconstitution. New stocks were received and reconstituted with filter-sterilized TE. Working stocks were made with filter-sterilized Nanopure H2O. All pipettes, tips, tubes, racks were UV-sterilized in the biological hood. The PCR reaction was set up in the biological hood. PCR set up is here. Used universal 16s bacteria primers (27F, 1492R). Sequences from Sara Kelly. Anneal 60C.

Lane 1 – 100bp ladder

Lane 2 – gDNA (5/13/2009)

Lane 3 – gDNA (5/26/2009)

Lane 4 – H2O

Lane 5 – H2O

Lane 6 – H2O

Lane 7 – H2O

Results: Still contamination in the water-only samples!!!

PCR – C.pugetti DNA from 20090513 & 20090526

This is a repeat of yesterday’s PCR with a fresh working stock in hopes of eliminating the source of contamination seen in the negative control yesterday.

Lane 1 – 100bp ladder

Lane 2 – 5/13 DNA

Lane 3 – 5/26 DNA

Lane 4 – Empty

Lane 5 – H2O

Results: Same problem as yesterday. Water sample is contaminated. This suggests that the primer stocks are contaminated. Likely due to the use of non-sterile, “old” TE for reconstitution of the primer stocks. Will re-order them and reconstitute them sterily.

PCR – C.pugetti DNA from 20090513 & 20090526

Performed PCR as set up here using universal bacterial 16s primers (sequences provided by Sara Kelly).

Lane 1 – 100bp ladder

Lane 2- 5/13 DNA

Lane 3 – 5/26 DNA

Lane 4 – H2O

Results: Contamination in the water sample. Hopefully this is just bad technique (never thought I’d say that about myself) and not general, bacterial contamination in the primer stocks, since the stocks (nor the PCR rxns) were prepared steriley. Will repeat.

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!