Continued MBD enrichment of C.virginica DNA from yesterday for Qiagen project.

Followed the MethylMiner Methylated DNA Enrichment Kit (Invitrogen) manufacturer’s protocol for input DNA amounts of 1 -10ug (I am using 8ug in each of two samples).

Since the protocol has two elution steps that are each saved separately from each other for each sample, I did the following to combine the two elution fractions into a single sample:

• Pelleted one elution fraction from each sample
• Discarded supernatant from pelleted sample
• Transferred second elution fraction to the pellet from the first elution fraction
• Pelleted second elution fraction

The rest of the ethanol precipitation procedure was followed per the manufacturer’s protocol.

Final pellets were resuspended in 25μL of Buffer EB (Qiagen) and stored temporarily on ice for quantification.

Continued MBD enrichment for C.virginica and Qiagen project from yesterday.

Followed the MethylMiner Methylated DNA Enrichment Kit (Invitrogen) manufacturer’s protocol for input DNA amounts of 1 -10ug (I am using 8ug in each of two samples).

Performed a single, high-salt elution.

Samples were precipitated O/N @ -80C.

As part of a project with Qiagen to have them try out some of our DNA with their newest DNA bisulfite conversion kit, I previously isolated DNA from Crassotrea virginica (Eastern oyster) and sheared to ~420bp.

Next, I needed to enrich the samples for methylated DNA. Did this using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen). Followed the manufacturer’s protocol for input DNA amounts of 1 -10ug (I am using 8ug in each of two samples). Below are the exact volumes used for various steps:

Made 1x Bind/Wash Buffer

• 2.88mL 5x Bind/Wash Buffer

• 720uL molecular biology grade H₂O

Beads:

• 80uL beads per sample

MBD-biotion protein:

• 56uL per sample

Diluted the two sheared DNA samples to 25ng/uL:

• C_iV_i = C_fV_f

• (58.4ng/uL)(136uL) = (25ng/uL)(V_f)

• V_f = 317.7

• Add 181.7uL H₂O to DNA to get 317.7ul (i.e. 25ng/uL)

Samples were incubated O/N in the 4C in the rotator.

We opted to go with ZymoResearch for this project because they could do the bisulfite treatment and finish the sequencing by the end of January.

Submitted the following 18 Ostrea lurida MBD-enriched gDNA samples to ZymoResearch for bisulfite treatment and subsequent Illumina sequencing (50bp, single read):

The samples will be bisulfite treated, Illumina libraries constructed, multiplexed, and this multiplexed library will be sequenced three times.

Enrichment was performed using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen) according to the manufacturer’s protocol with the following changes:

– Used 25uL of Dynabeads M-280 (10uL/ug of input DNA) and 15uL of MBD-Biotin Protein (7uL/ug of input DNA).

– Followed the corresponding instructions for the volumes listed above and for quantities of input DNA > 1ug – 10ug

– A single elution with 2000mM NaCl was performed

– EtOH precipitation: Samples were incubated over the long weekend at -80C.