Tag Archives: restriction digestion

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.

Restriction Digest – Oly gDNA for RAD-seq w/AlfI

The previous attempt at making these RAD libraries failed during the prep-scale PCR, likely due to a discrepancy in the version of the Meyer Lab protocol I was following, so I have to start at the beginning to try to make these libraries again.

Since the input DNA is so degraded, I’ve repeated this using 9μg of input DNA (instead of the recommended 1.2μg). This should increase the number of available cleavage sites for AlfI, thus improving the number of available ligation sites for the adaptors.

Used a subset (10 samples) from the Ostrea lurida gDNA isolated 20150916 to prepare RAD libraries.

Followed the 2bRAD protocol (PDF) developed by Eli Meyer’s lab.

Prepared 9.0μg of each of the following samples in a volume of 9.5μL:

Google Sheet: 20151028_RADseq_DNA_calcs

 

 

Prepared master mix for restriction enzyme reaction:

REAGENT SINGLE REACTION (μL) x11
DNA 9.5 NA
10x Buffer R 1.2μL 13.2μL
150μM SAM 0.8μL 8.8μL
AlfI 0.5μL 5.5μL

 

Combined 2.5μL of the master mix with 9.5μL of each DNA sample in 0.5mL snap cap tubes. Incubated @ 37C O/N in thermal cycler (PTC-200; no heated lid).

Restriction Digest – Oly gDNA for RAD-seq w/AlfI

Previously initiated the RAD-seq procedure for the sample set described below. However, the test scale PCR yielded poor results. Katherine Silliman suggested that the poor performance of the test scale PCR was likely due to low numbers of adaptor-ligated fragments. Since the input DNA is so degraded, I’ve repeated this using 9μg of input DNA (instead of the recommended 1.2μg). This should increase the number of available cleavage sites for AlfI, thus improving the number of available ligation sites for the adaptors.

Used a subset (10 samples) from the Ostrea lurida gDNA isolated 20150916 to prepare RAD libraries.

Followed the 2bRAD protocol (PDF) developed by Eli Meyer’s lab.

Prepared 9.0μg of each of the following samples in a volume of 10μL:

Google Sheet: 20151009_RADseq_DNA_calcs

 

Prepared a 150μM working stock of the SAM buffer needed for the restriction digestion by diluting 30μL of the supplied stock (500μM) in 70μL NanoPure H2O (total volume = 100μL). This working stock was stored @ -20C in FTR 209 in the “RAD-seq Reagents” box.

Prepared master mix for restriction enzyme reaction:

REAGENT SINGLE REACTION (μL) x11
DNA 8 NA
10x Buffer R 1.2μL 13.2μL
150μM SAM 0.8μL 8.8μL
AlfI 0.5μL 5.5μL
H2O 1.5μL 16.5μL

 

Combined 4μL of the master mix with 8μL of each sample in 0.5mL snap cap tubes. Incubated @ 37C 2hrs. in thermal cycler (PTC-200; no heated lid). Heat inactivated the digest @ 65C for 10mins.

Restriction Digest – Oly gDNA for RAD-seq w/AlfI

Used a subset (10 samples) from the Ostrea lurida gDNA isolated 20150916 to prepare RAD libraries. This will be done to assess whether or not these samples, which appear to be heavily degraded, are viable for RAD-seq.

Followed the 2bRAD protocol (PDF) developed by Eli Meyer’s lab.

Prepared 1.2μg of each of the following samples in a volume of 10μL:

Google Sheet: 20150930_RADseq_DNA_calcs

 

Prepared a 150μM working stock of the SAM buffer needed for the restriction digestion by diluting 30μL of the supplied stock (500μM) in 70μL NanoPure H2O (total volume = 100μL). This working stock was stored @ -20C in FTR 209 in the “RAD-seq Reagents” box.

Prepared master mix for restriction enzyme reaction:

REAGENT SINGLE REACTION (μL) x11
DNA 8 NA
10x Buffer R 1.2μL 13.2μL
150μM SAM 0.8μL 8.8μL
AlfI 0.5μL 5.5μL
H2O 1.5μL 16.5μL

 

Combined 4μL of the master mix with 8μL of each sample in 0.5mL snap cap tubes. Incubated @ 37C O/N in thermal cycler (no heated lid).

Restriction Digest – Oly Oyster gDNA-01 for RAD Sequencing (from 20141029)

Samples required two days of drying for all samples to fully dry down.

Reconstituted all samples in 20uL of PCR H2O.

Performed restriction digests:

Incubated at 37C for 90mins and then inactivated enzyme @ 80C for 20mins. Incubation was done in thermal cycler using heated lid. Plate was stored @ -20C.

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.

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.