Tag Archives: MspI

Samples Submitted – C. virginica gDNA, MBD, and MspI to Qiagen

Sent Crassostrea virginica samples to Qiagen, as part of the collaboration we have with them for testing their new bisulfite conversion kit on various reduced representation DNA.

Samples were sent on dry ice via FedEx International Priority: 771231112481

Here are the samples I sent:

gDNA C.virginica – Genomic DNA, 20uL, 58.4ng/uL
MBD 1 virginica – Fragmented (~400bp average size), MBD-enriched, 25uL, 18.3ng/uL
MBD 2 virginica – Fragmented (~400bp average size), MBD-enriched, 25uL, 19.6ng/uL
MspI 1 virginica – gDNA digested with MspI, 25uL, 53.4ng/uL
MspI 2 virginica – gDNA digested with MspI, 25uL, 31.0ng/uL

Genomic DNA was isolated from mantle tissue using the E.Z.N.A. Mollusc DNA Kit (Omega). DNA was eluted with the Elution Buffer supplied with the kit.

MBD enrichment was performed using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen). DNA was resuspended in Buffer EB (Qiagen).

MspI digestions were performed using MspI (NEB) and were subjected to a phenol:chloroform cleanup, post-digestion. DNA was resuspended in Buffer EB (Qiagen).

No tests have been performed on the samples to evaluate the presence/absence of non-oyster DNA.

Reference genome is available here: https://www.ncbi.nlm.nih.gov/assembly/GCF_002022765.2/

Notebook entries:

gDNA Isolation:

http://onsnetwork.org/kubu4/2017/12/11/dna-isolation-quantification-crassotrea-virginica-mantle-gdna/

MBD:

DNA Shearing and Bioanalyzer assessments: http://onsnetwork.org/kubu4/2017/12/11/dna-sonication-bioanalzyer-c-virginica-gdna-for-medip/
Day 1: http://onsnetwork.org/kubu4/2018/01/08/mbd-enrichment-crassostrea-virginica-sheared-dna-day-1/
Day 2: http://onsnetwork.org/kubu4/2018/01/09/mbd-enrichment-crassostrea-virginica-sheared-dna-day-2/
Day 3: http://onsnetwork.org/kubu4/2018/01/10/mbd-enrichment-crassostrea-virginica-sheared-dna-day-3/
Quants: http://onsnetwork.org/kubu4/2018/01/10/dna-quantification-c-virginica-mbd-enriched-dna/

MspI Digestion:

Digestion: http://onsnetwork.org/kubu4/2018/01/11/restriction-digestion-mspi-on-crassotrea-virginica-gdna/
Phenol:Chloroform cleanup: http://onsnetwork.org/kubu4/2018/01/11/phenolchloroform-extractions-and-etoh-precipitations-mspi-digestions-of-c-virginica-dna-from-earlier-today/
Quants: http://onsnetwork.org/kubu4/2018/01/11/dna-quantification-mspi-digested-crassostrea-virginica-gdna/

Restriction Digestion – MspI on Crassotrea virginica gDNA

Digested two 1.5μg aliquots of Crassostrea virginica isolated 20171211, as part of the project we’re doing with Qiagen.

Digestion reactions:

Component Volume(μL)
DNA (1.5μg) 25.7
10x CutSmart Buffer (NEB) 5.0
Water 17.3
MspI (NEB) 2
TOTAL 50

MspI info:

  • NEB R0106T (100,000U/mL; rec’d 20171214)

Reactions were carried out in 0.5mL snap-cap PCR tubes and incubated for 15mins @ 37oC in a PTC-200 thermalcycler (MJ Research), no heated lid.

Samples will be subjected to a phenol:chloroform extraction for cleanup.

EtOH Precipitations – HpaII and MspI 2nd Round Digests from 20101124

Samples were EtOH precipitated, according to protocol. Samples were resuspended in 20uL of Qiagen’s EB and spec’d.

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

Results:

Spreadsheet of spec values is here. Overall, poor recoveries from all of the digested samples, but decent recoveries from the undigested samples. The samples were passed to Mac who performed qPCR using two different primer sets. Please see her notebook for the results of the qPCR.

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.

Restriction Digests – Various gigas gDNAs of Mac’s

Performed restriction digests. Made dilutions of all DNAs involved of 25ng/uL. Made enough for a total of 9 digests could be performed on each DNA. This allowed using 10uL of each DNA for each rxn, more mileage out of the lowest concentration sample (R37-01), and allowed for the use of master mixes when preparing the digests. All calculations/dilutions/master mixes can be seen here. Each DNA was digested individually with HpaII, MspI and undigested. Incubated the digests 4hrs @ 37C. After digestion, performed an EtOH precipitation. Added 0.1 vols of 3M NaOAc (pH=5.2), then 2.5 vols of 100% EtOH. Mixed by inversion and incubated 30mins @ -20C. Pelleted DNA 16,000g, 30mins @ 4C. Discarded supe. Washed pellets with 1mL 70% EtOH. Pelleted DNA 16,000g, 15mins, 4C. Discarded supe. Resuspended DNA in 10uL PCR H2O and spec’d.

Results:

Well, the recovery of DNA is very low. The best recovery is ~50% while the worst is around ~1%.

I did not proceed with the intended qPCR due to the low yields and the fact that I don’t know if we’ve previously tested how sensitive our assay(s) our for our target genes. Will discuss with Steven/Mac next week.