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 – Test QT Kit with No RT Abalone rxns from 20090408

Anneal 55C. PCR set up is here (bottom half of sheet).

Lane 1 – Hyperladder

Lane 2 – gDNA

Lane 3 – cDNA pool

Lane 4 – No RT 06:5-31

Lane 5 – No RT 06:6-43

Lane 6 – No RT 08:3-5

Lane 7 – No RT 08:3-6

Lane 8 – No RT 08:3-7

Lane 9 – H2O

Lane 10 – H2O

Lane 11 – 100bp ladder

Results: No signal in the gDNA. Appropriate sized band in cDNA pool. Nothing in the water samples. HOWEVER, got bands in tow of the “No RT” rxns (08:3-6/7)!! It’s odd that the gDNA didn’t produce any signal, but there shouldn’t be any signal in the No RT rxns. This indicates gDNA contamination. Should have also tested RNA and DNased RNA. Will test these in order to determine if that system is better at eliminating gDNA carryover.

PCR – Bay/Sea scallop gDNAs

Used higher annealing temps to improve primer specificity, compared to yesterday’s results. PCR set up and plate layout is here.

See the PCR/plate set up link for samples. Hyperladder is placed between every 12 samples.

Results: See this Google Spreadsheet for a summary of the 4 gels from the last two days.

PCR – Bay/Sea scallop gDNA isolated earlier today

Used 3 sets of reverse primers:

Bay_Actin_Rv0

Bay_Actin_Rv2

Sea_Actin_Rv2

Primers were slected based on information from Steven’s notebook (#8, 12/30/2007-1/3/2008). Anneal temp 53C.

PCR set up here . Plate layout here .

Samples were run out by Steven the following day.

Gel 1 of 3

Gel 2 of 3

Gel 3 of 3

Results:

PCR – Abalone gDNA/RNA/cDNA w/new TOLLIP primer

Performed a new PCR on the three types of samples listed above with new TOLLIP primers. The new TOLLIP primers (H.discus_806_F/Ab_866_Rv) surround a putative intron. Thus, they will be useful for determining the presence of gDNA contamination in RNA/cDNA. Anneal temp 55C. PCR set up is here .

Lane 1 – Hyperladder

Lane 2 – gDNA

Lane 3 – cDNA (QT)

Lane 4 – RNA (untreated)

Lane 5 – DNased RNA

Lane 6 – QT Kit, No RT

Lane 7 – H2O

Lane 8 – H2O

Results: This could possibly be the most confusing gel I’ve ever had the “pleasure” of running/analyzing, despite that it only has 7 samples.

No band in the gDNA sample, which could be explained by the intron size being too large for amplification with a basic polymerase. The cDNA worked as expected and contains a band of ~150bp. The RNA sample has a faint band of ~750bp. The DNased RNA sample has a band of ~400bp. The differences seen betweeen the gDNA (Lane 2), RNA (Lane 4) and DNased RNA (Lane 5) are truly bizarre. The No RT sample has no band. And, to top things off, one of the H2O samples is blank , but the other one has a band of ~1500bp! Ugh. How is all of this even possible?