Restriction Digestions – HpaII and MspI on Mac’s C.gigas Samples: Round 2

Continued with 2nd round of digestions from yesterday. All samples were resuspended in 25uL of H2O yesterday, so brought volume up to 44uL with H2O, added 5uL of appropriate 10X Buffer (HpaII = NEB Buffer #4, MspI = NEB Buffer #1), added 1uL of enzyme, incubated 37C for 3hrs. Heat-inactivated all samples @ -80C for 30 mins.

Phenol:Chloroform Extractions

Restriction digests  were mixed with equal volume (50uL) of phenol:chloroform:IAA (25:24:1) and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to a clean tube and an equal volume (50uL) of chloroform was added. Samples were mixed and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to clean tubes and stored @ -20C. Will EtOH precipitate and spec on Monday.

Phenol:Chloroform Extractions and EtOH Precipitations – HapII and MspI digests from yesterday

Restriction digests from yesterday were mixed with equal volume (50uL) of phenol:chloroform:IAA (25:24:1) and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to a clean tube and an equal volume (50uL) of chloroform was added. Samples were mixed and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to clean tubes and EtOH precipitated, according to protocol. Samples were resuspended in 25uL of H2O and spec’d.

Samples are labeled as Parent (P), #, tissue, enzyme (MspI = M, HpaII = H, Undigested = U)

Results:

Here is a link to a spreadsheet with ODs. Average recovery was ~734ng, which is only a 36% recovery (started with 2000ng). Will need to discuss with Mac and Steven to see if it’s worth continuing with these sample through a second round of digests/phenol:chloroform extraction/EtOH precipitation, as I don’t know what quantity of DNA is required/desired for the subsequent methylation specific PCR (MSP), OR if I should/need to perform a repeat of these 1st-round digestions in order to end up with sufficient DNA for MSP.

Restriction Digestions – HpaII and MspI on Mac’s C.gigas gDNA Samples: Round 1

Set up restriction digests for subsequent analysis by methylation specific PCR (MSP). This will be the first of two rounds of digestion with the same enzyme on each sample. Samples and master mixes are here. Samples were incubated 3hr. @ 37C. All samples were heat inactivated at 80C for 30mins and then stored @ -20C.

QPX Washes

Washed 4 day old QPX cultures. 3 isolates (S-1, TD-81, ATCC), two flasks (13mL) of each were washed in the following manner:

  1. Each flask’s contents were transferred to a 50mL conical tube.

  2. Each tube was topped with sterile sea water and mixed by inversion numerous times.

  3. Tubes were centrifuged 10mins, 3000g at RT.

  4. Transferred mucus and pellet to empty 50mL conical tube.

  5. Topped with sterile sea water and mixed by inversion numerous times.

  6. Centrifuged 10mins, 3000g at RT.

  7. Removed as much supe/mucus as possible without disturbing the pellet.

  8. Topped with sterile sea water and mixed by inversion numerous times.

  9. Repeat steps 7 through 8 until sample is mucous-free and the pellet can be resuspended.

Pellets were eventually resuspended in 1mL of sterile sea water, split between two 1.5mL snap cap tubes and then brought up to 1mL with sterile sea water.

gDNA Isolation

Isolated gDNA from gray whale skin, human cheek cells (my own!) and two different species of algae (species 1, species 2) using Qiagen’s DNEasy Blood & Tissue Kit according to protocol. Incubated all samples at 55C for 1hr. Eluted DNA with 50uL of Buffer AE. Spec’d samples on NanoDrop 1000.

Cheek cells were scraped from the inside of my cheek with a sterile toothpick. The toothpick was transferred to a 1.5mL snap cap tube containing the appropriate buffer. The tube was vortexed to help dislodge cells from the toothpick. The toothpick was then removed and the sample treated according to protocol.

1mL of algae cells were collected from each liquid culture, cells were pelleted by spinning 16,000g for 1min @ RT, supe removed, 180uL of Buffer ATL added and then vortexed to dislodge/break up pellet. Sample was treated according to protocol.

Results:

Well, no detectable quantities of DNA in 3 of the 4 samples. There appears to be something in the gray whale skin gDNA extraction, however the OD260/280 ratio is just crazy, leading me to believe that there’s not really any usable DNA present. Will give gray whale gDNA sample to Caroline for class and will talk with Steven and Caroline concerning their interest in performing another quick extraction on more algae to use for class this afternoon.

EtOH Precipitation – Whale gDNA from 20101022

Precipitated whale gDNA in hopes of producing a sample with a higher concentration. Added 0.1 vols of 3M NaOAc (10uL), 2.5 vols of 100% EtOH (275uL), mixed thoroughly and incubated @ -20C for 1hr. DNA was pelleted by spinning sample @ 16,000g, 30mins, 4C. No visible pellet. Supe was removed, sample was washed with 1mL 75% EtOH, and pelleted by spinning @ 16,000g, 15mins, 4C. Supe was removed, sample resuspended in 10uL nuclease-free H2O and spec’d.

Results:

No DNA to speak of (spec data not shown).

gDNA Isolation

Isolated gDNA from crab (unknown species), starfish exposed to RoundUp (unknown species) and gray whale blubber using Qiagen’s DNEasy Kit, according to manufacturer’s protocol. Tissue was incubated at 55C with Proteinase K for 1hr. gDNA was eluted with 100uL of Buffer AE and spec’d.

Results:

Gray whale sample has virtually no DNA. Will try to precipitate the whale gDNA in order to obtain a more concentrated sample. The other two samples look good, with good yields and good OD260/280 ratios.

Received Hard Clam Samples and Live Clams from MBL

Received 82 gill samples in RNA Later in 3 microfuge tube racks from MBL (Scott Lindell). Samples were catalogued, boxed (1 box) and stored at -80C.

The live clams were received in two bags one containing ~12 labelled MA4 and one containing ~12 labelled BX1. Clams were NOT counted and the quantity may be different. Clams were temporarily stored @ 4C.

Received Hard Clam Samples from Rutgers

30 gill tissue samples in RNA Later from CA, MA, & MAX each.

30 hemolymph samples (in RNA Later?) from CA, MA, & MAX each.

Presumably these are from the same individuals. Tubes were boxed (a total of 3 boxes), labeled and stored @ -80C.

Here is a note included from Emily with the samples .

qPCR – Test Plate for Opticon 2

Ran a full plate for testing well-to-well consistency (or, inconsistency!) of the Opticon 2, since it’s been behaving poorly lately. This will provide us with an idea of whether or not the oddities that we’ve been witnessing have any effect on our actual data.

Used C.gigas gDNA (DH15 from 20100519; 0.5128ug/uL) and IL17 Internal Fw/Rv primers (SR ID: 255, 256), which have previously produced an amplicon with gDNA. Master mix calcs/plate layout/cycling parameters/etc are here.

DNA was combined in master mix so that all wells received ~100ng of gDNA.

Results: