15 April 2014


ddRADseq and restriction enzymes

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ddRADseq trial 2

CCThermBe

Heat shocked ‘blue’ ants.

ddRADseq

Started trial ddRADseq library prep using 12 expendable Aphaenogaster samples that Nicole extracted last week - 3 ants from each colony.

Measured concentration using Qubit.

Sample Colony ng/mL Total DNA (ng)
1 EW205 424 1696
2 EW110 >600 2400
3 2012A1 291 1164
4 DG23 580 2320
5 20130818-Mike 349 1396
6 EW20 QR 352 1408


Total DNA = Qubit sample ng/mL * 100 * (1/1000 ul) * 40 ul

Combined Total DNA = 8 ug

For ‘real’ library would pool 24 - 48 samples, here only using 6 so used all DNA from each sample to ensure large volume of DNA in pooled sample for size selection and purification steps.

Double digest

50 ul rxn volume following NEB protocol

Reagent Volume (ul)
NlaIII 1
MluCl 1
DNA 40
CutSmart Buffer 5
H2O 4


3 hr digestion at 37°C.

Included “C” Control sample of dH2O only.

Sent 2ul aliquot of each sample for Bioanalyer analysis of cutting efficiency.



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