qPCR was performed with 16s_sybr primers on a subset of the “No RT” cDNA rxns from yesterday at both 55C and 60C. Sample set up and plate layout is here.
Results: Still getting signals in the “No RT” rxns and possibly in the waters. Cut run short to start another. Will test Lisa’s previously DNased RNA.
qPCR was performed with 16s_sybr primers on a subset of the cDNA rxns and all of the “No RT” rxns from earlier to detect the presence of contaminating gDNA. qPCR was also performed with the Rab7_sybr primers on a subset of the cDNA rxns to check that the assay would work. The qPCR set up sheet/plate layout is here. Annealing temp = 55C.
Results: Apparently the gDNA wipeout step did NOT work! Also, some signal in the water samples.
cDNA was made from the above RNA samples using the Qiagen Quantitect RT Kit. The samples were laid out in a PCR plate. 274.2ng of RNA was used in the rxn for each sample, based on the lowest concentration RNA sample (08:3-20) to equalize all the samples. The Genomic Wipeout step of the kit requires 2uL of Genomic Wipeout enzyme/buffer to be added to 12uL of an RNA sample, so the calculations were done and can be found here. A check mark on the calculation sheet indicates that the water and then the RNA was added to the appropriate wells. Those with two check marks were used for a “No RT” rxn and thus, have duplicate wells (see plate layout).
The plate was mixed, spot spun, uncubated at 42C for 2mins and immediately placed on ice.
The RT and No RT master mixes were set up on ice and then added to the respective wells (see sheet here).
UPDATE: cDNA plate was discarded 20120320 by SJW.
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).
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.
Abalone RNA isolated from this week was qPCR’s with 16s primers to determine if gDNA contamination was present. PCR/plate layout is here.
Results: All samples appear to have gDNA contamination. Will use Qiagen QT Kit for cDNA.
gDNA was isolated from the following using the Qiagen DNEasy Kit:
xCvC-19t (3/26/2009)
xCvE-13t (3/16/2009)
xCvC-17t (3/18/2009)
UNTc-1.5t (3/18/2009)
VATm-1.2t (3/16/2009)
xCvC-11t (3/18/2009)
BC05Ca18c/H5 (3/27/2009)
xCvC-12t (3/16/2009)
500uL was of each sample was transferred to a 1.5mL snap cap tube. The samples were pelleted by spinning 5 mins @ 16,000g and washed 2x w/ 1x PBS pH=7.6. Samples were then processed according to Qiagen protocol. Initial digestion with Proteinase K was 2hrs.
Total RNA was isolated from the following abalone digestive gland samples using the RNA Powersoil Kit, according to their protocol:
08:3-17
08:3-18
08:4-2
08:4-3
08:4-4
08:4-5
08:4-7
08:4-8
08:4-11
Notes: 08:4-5 took hours to wash the column. 08:4-4 ended up with a dark RNA pellet and the reuspended RNA is discolored (brown-ish).
Results: All the RNA looks good except for 08:4-4, but that isn’t surprising (see note above). Samples were stored @ -80C in the boxes from which the tissue samples came.
Total RNA was isolated from the following abalone digestive gland samples using the RNA Powersoil Kit, according to their protocol:
08:3-6
08:3-7
08:3-8
08:3-9
08:3-14
08:3-15
08:3-16
08:3-24
Results: RNA looks great. Stored @ -80C in box where samples came from.
Total RNA was isolated from the following abalone digestive gland samples using the RNA Powersoil Kit, according to their protocol:
08:3-11
08:3-12
08:3-13
08:3-19
08:3-20
08:3-21
08:3-22
08:3-23
Notes: After phase separation, 3-11 and 3-12 had milky/cloudy aqueous phases. These two samples were subjected to another 10min spin @ 2500g, but this spin made no difference in their appearance. Other samples were clear or slightly translucent at worst.
Results: RNA looks great in nearly all of the samples. RNA has been stored @ -80C in the same box from where the tissue was taken.
