Tim's+Notebook

Run qRT-PCR - Juvenile gill cDNA P450 - run file: 20090925_095109
 * 25th September - Friday**

Run qRT-PCR - Juvenile gill cDNA TNF - run file: 20090924_202723 HiF1a - run file: 20090924_164550 Interleukin - run file: 20090924_123556
 * 24th September - Thursday**

Run qRT-PCR - juvenile gill cDNA Prostaglandin - run file:20090923_133453 Prx6 - run file: 20090923_094226
 * 23rd September - Wednesday**

Run qRT-PCR - juvenile gill cDNA IkB run file:20090922_171022 Elongation factor1 run file:20090922141956
 * 22nd September - Tuesday**

qRT-PCR reaction Reagent per RXN 67 RXN Immunomix 12.5 837.5 SYBR13 (50 uM) 1 67 primer f (10mM) 0.5 33.5 primer R (10mM) 0.5 33.5 dH2O 5.5 368.5 Template (dilute 1:4) 5.0 N/A

PCR parameters: 95C 10min 95C 15s 55C 15s 45 cycles 72C 30S Melt curve analysis 21C 10 min Finish

Reverse Transcription Reaction


 * 21st September - Monday**


 * 20th September**

DNAse treat remaining adult total RNA samples following manufacturers protocol Sample ng/ul 260/280 260/230 AC1 379 1.83 .81 AC2 636 1.51 .31 AC3 407 1.83 .92 AC4 671 1.42 .33 AC5 333 1.69 .68 AC6 672 1.38 .34 AC7 454 1.90 1.15 AC8 395 1.76 .86 AV1 458 1.82 1.03 AV2 867 1.51 .35 AV3 1048 1.5 .38 AV4 1094 1.54 .39 AV5 722 1.47 .33 AV6 927 1.45 .36 AV7 830 1.44 .35 AV8 1253 1.48 .42 CC1 668 1.43 .33 CC2 424 1.8 .99 CC3 364 1.70 .77 CC4 404 1.79 .91 CC5 355 1.63 .75 CC6 588 1.4 .32 CC7 423 1.82 .97 CC8 623.6 1.4 .33 CV1 748 1.45 .35 CV2 374 1.82 .82 CV3 590 1.43 .31 CV4 726 1.54 .31 CV5 361 1.76 .78 CV6 338 1.74 .69 CV7 318 1.72 .65 CV8 360.7 1.77 .76

Perform RT reaction Step 1 Reagent per RXN 40RXN dNTPs .5 20 Primer * .5 20 RNA sample 5.0

Incubate samples at 70C for 5 min, on ice 1min
 * 50/50 oligodTs and random primers

Step 2 Reagent per RXN 35 RXN 5xbuffer 2 70 RT .5 17.5 dH2O 1.5 52.5

PCR RTRXN in sam's list.

Dilute cDNA with 90ul dH2O water (1/10 dilution).

Perfrom qRT-PCR with elongation factor1 primers Reagent per RXN 68RXN Immunomix 12.5 850 SYBR13 1.0 68 Forward .5 34 Reverse .5 34 dH2O 5.5 374 Template 5

PCR parameters: 95 10 min 95C 15s 55C 15s 45 cycles 72C 30s Melt Curve 21C 10min

qPCR didnt work. Some samples did amplify after 40 cycles, so the master mix etc was ok. What am I stuffing up. I think it must be the reverse transcription. Expression that I measured earlier in the week was most likely gDNA contamination of the samples. Explain why I saw no significant differences in IKB expression btw controls and vibrio samples. Email Steven and Sam.

Total RNA purification of juvenile oyster samples following Tri-reagent Protocol.

Measure purity and quantity of total RNA of juvenile samples before DNAse treatment

Sample ng/ul 260/280 260/230 AC1 AC2

STOP - I AM OBVIOSULY STUFFING SOMETHING UP. PLAN IS TO SPEAK TO STEVEN AND SAM PREFOR I RESUME WORK.

18th Sep 09 Perform qPCR using 18S primer pair on total RNA samples that have been treated twice with DNAse. 1ul of total RNA diluted in 50ul of dH2O

Reagent per RXN 18RXN Immunomix 12.5 225 dH2O 5.5 99 18S forward 0.5 9 18S Reverse 0.5 9 Template 5.0

No genomic DNA detected in samples using 40 cycles

Using the Turbo DNA-free Kit (applied biosystems), a second round of DNAse treatment was perform on adult hemocyte total RNA from air+control samples. Followed rigorous DNase treatment protocol.

Total RNA purity and quantity measure using ND-1000.

Sample ng/ul 260/280 260/230 AC1 379 1.83 .81 AC2 636 1.51 .31 AC3 407 1.83 .92 AC4 671 1.42 .33 AC5 333 1.69 .68 AC6 672 1.38 .34 AC7 454 1.90 1.15 AC8 395 1.76 .86

17th Sep 09 Phenoloxidase assay

16th Sep 09

15th Sep 09

14th Sep 09 Purified total RNA from adult PO hemocytes using tri-reagent (molecular research centre, INC) following abbreviated protocol. Total RNA was resuspended in 20ul dh20 and DNAse treated with Turbo DNA-freeTM kit (applied biosystems) following manufacturers protocol. Total RNA frozen overnight at -80C.

9th Sep 09
 * Determine primer amplification efficiency.**

8th Sep 09
 * Optimised Primer Concentrations for qRT-PCR.**

Adult and juvenile PO challenged with 4.5x10exp5 CFU.ml of V. tubiashi for 8 h in 3 L aerated static aquariums at 15C. Control oysters were held in identical aquaria minus V. tubiashi. Hemocyte and gill samples were taken and frozen at -80C.
 * 7th Sep 09**

V. tubiashi grown overnight in 500 ml of LB broth + 2% NaCl at 150 rpm and RT.
 * 6th Sep 09**

2nd Sep 09 Fed oysters 5 ml of shellfish diet to each tank. Measure pH of tanks Tank pH Air 7.63 CO2 7.42

Reverse Transcription of RNA using Promega kit. Use 50/50 mix of oligo dTs and random primers Reagent per RXN 9 RXN RNA 3 N/A dNTPs 0.5 4.5 Primer 0.5 4.5

Heat samples at 70C for 5 min, cool on ice for 1 min.

Reagent per RXN 9 RXN 5xBuffer 2 18 RT enzyme 0.5 4.5 dH2O 3.5 31.5

Heat samples at 37C for 1 h, 95C for 15 min to inactivate enzyme, 4C hold.

Dilute cDNA by 90 ul. Freeze at -20C

Start CO2 bubbling on oyster stanks
 * 1st Sep 09**

Purified total RNA from hemocytes of PO that were part of Herschel's experiment looking at CO2 on oysters.

Total RNA was purified using the TRI Reagent method. Briefly, hemocytes (frozen -80C) were homogenised in 1 mL of TRI Reagent for 5 min at RT. 200 ul of chloroform:isoamyl:alcohol was added to each tube and vortex. Tubes centrifuged at 15 000 rmp for 15 min at 4C. Supernatant transferred to new tube and 500 ul of isopropanol added, vortexed and centrifuged at 15 000 rpm for 8 min. RNA pellet was washed in 1 ml of 75% etoh and pellet resuspended in 20 ul of dH2O. Quantity of total RNA determined using a spec (ND-1000).

Sample ng/ul 260/280 260/230 pH A 792 1.43 0.45 pH B 1037 1.65 0.6 pH C 936 1.47 0.5 pH D 440.3 1.61 0.89

Cont A 698 1.42 0.4 Cont B 763 1.56 0.45 Cont C 1126 1.55 0.56 Cont D 652.1 1.53 0.49

Genomic DNA contamination removed using the Turbo DNA-freeTM Kit (applied biosystems).

Step 1 Reagent per RXN (ul) 10 RXN (ul) RNA sample 20 N/A Turbo Buffer 2.5 25 DNase 0.5 5.0 Step 2 Water bath (37C) for 30 min Step 3 Add 2.5 ul of DNase Inactivation Reagent to each tube and mix for 3 min Step 4 Centrifuge 15 000 rpm for 2 min and transferred supernatant to new tube. Determine total RNA by spec (ND-1000)

Sample ng/ul 260/280 ul to 1000ng ul to 3 ul pH A 454 1.89 2.2 0.8 pH B 441 1.88 2.3 .7 pH C 413 1.73 2.4 0.6 pH D 334.9 1.61 3.0

Cont A 443 1.88 2.25 .75 Cont B 431 1.85 2.3 0.7 Cont C 618 1.44 1.7 1.3 Cont D 318 1.68 3.0

31st Aug 09 Feed oyster with 5mL of shellfish diet 1800 to each tank

28th Aug 09 Repeat 16S PCR as below except using new aliquots of 27F and 1492R.

To confirm isolate, perform 16S PCR using 27F and 1492R bacterial universal primers (Lane, 1991). Pick one colony into 1 mL of dH20 and boil for 10 min at 95C. Spin tube at 10 000 rpm for 5 min. Transfer supernatent to new tube, dilute 1/10 and 1/100.
 * 27th Aug 09**

dH20 10.5 52.5 Taq Master Mix 12.5 62.5 27F 0.5 2.5 1492R 0.5 2.5 Template 1.0
 * Reagent** **per RXN 5 RXN**

PCR Parameter Step 1 95C for 10min Step 2 95C for 15 sec Step 3 48C for 1 min Step 4 72C for 3 min Repeat step2-4 35 times Step 5 72C for 10 min Step 6 hold 4C

Seperate PCR products by electrophoresis using 1.0% agarose/EtBr gel. PCR worked to well - Positive band in Negative Control. Suspect bacterial contamination of primers as all other reagents/tubes/tips were freshly autoclaved.


 * 26th Aug 09**

Plated V. tubiashi (strain X001-12-1) onto LB agar + 2%NaCl