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.
Set up qPCR using the following Virginica primers provided by Mac:
Cv_BgBL
Cv_BGBP
Cv_CysB
Cv_HMG
Cv_HSP70
Here is the qPCR workup sheet .
Submitted four samples (4uL of each) for Agilent Bioanlyzer:
SR01 = total RNA
SR02 = Ambion kit x 1
SR03 = Ambion kit x 2
SR04 = PolyA Tract kit
Results:
Precipitation was continued from yesterday. Samples were resuspended in 25uL of The RNA Storage Solution (from PolyAPursit Kit) and spec’d. Samples were stored @ -80C in Sam’s RNA box.
DNased RNA from earlier today was split into four equal parts (175uL = 39.8ug). Three will be used for mRNA isolation and the fourth will remain as total RNA. Three of these were precipitated according to Ambion PolyAPurist Protocol: 1/10 volume 5M ammonium acetate, 1uL glycogen and 2.5 volumes of 100% EtOH. Incubated @ -80C for 30 mins. One sample was processed with the Promega PolyA Tract kit. The remaining two samples were processed according to PolyAPurist Protocol. Of those two, one of the samples was processed a second time to evaluate the effectiveness of running a sample through the PolyAPurist Protocol twice.
mRNA samples were precipitated O/N @ -20C according to the PolyAPurist Protocol.
DNAsed RNA using Ambion Turbo DNA-free kit, following the rigorous procedure. Diluted total RNA to 0.2ug/uL (Vf = 720uL). Added 1uL DNase and incubated the tube @ 37C for 30mins. Added an additional 1uL DNase and continued incubated for 30mins. Added 0.2 volumes of DNase Inactivation Reagent (158.4uL) and incubated at RT for 10mins with periodic mixing. Pelleted inactivation reagent according to protocol and transferred supe (DNA-free RNA) to clean tube.
Results: RNA looks really nice. Have a large quantity of RNA (700uL x 0.2275ug/uL = 159.25ug). Will split into four equal parts and isolate mRNA from 3 of the 4. Those three will be:
Ambion kit x 1
Ambion kit x 2
Promega PolyA Tract kit.
This is an exact repeat of the qPCR from earlier today, but using a fresh working stock of the Vtub_16s_V3 primers. The plate layout/qPCR workup is here.
Results: Same as earlier today. Must be a bacterial contaminant somehwere that these 16s primers are picking up. Will order IGS primers that are species specific found in Lee et al. 2002.
