5Trout Experimental Prep

One library from the previous page will be prepped in an alternate way. Digestion and Ligation will be done the same. But, a portion from each well at this stage will be pooled in larger pools, size selected, and then PCRed.

Plates MPR1, MPR2, and YCT6:

After Ligation and before dilution, 2 x 2 ul was taken from each resultant ligation and combined in two of four variant pools for each plate(The first 6 column, the last 6 columns, odd columns, even columns). Then, these 12 pools were size selected for 300-366 bp.

If need be, these will then be PCRed minimally.

PCR Amplification

Reagent	ul/rxn	rxns	ul needed (x1.4)

Reagent	ul/rxn	rxns	ul needed (x1.4)
H2O	9.52	350	162
5x iProof buffer	4	350	1300
10 mM dNTPs	0.4	350	130
50 mM MgCl2	0.4	350	130
5 uM Illumina Primers	1.33	350	432
iProof TAQ	0.2	350	65
DMSO	0.15	350	49
total	16	350	5200

Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel

Add 4uL of template from pooled plates with Benchsmart

Seal, Vortex, Spin down, and then run on Thermocycler program: GBS2:

Temp C	Cycles	Time

Temp C	Cycles	Time
95*	1X	3:00
98	12X	0:30
60	12X	0:30
72	12X	0:40
72	1X	10:00
4*	1X*	0:00*

Fine Tuning

TapeStation looked good showing ~2nM product. Unfortunately, it also shows the primers. Will do a bead cleanup and check again.

Initial TapeStation:

Bead Cleaned TapeStation:

Run Final Product on qPCR for check and quant

Result from qPCR check :

Sample Name	qPCR	Sample ul	RSB ul

Sample Name	qPCR	Sample ul	RSB ul
Exp5Trout1	1.86 nM	53.8	46.2
5Trout1Org	3.05 nM	32.8	67.2

load =800 pM

80 ul 1 nM stock, 4 ul 1 nM PhiX, 16 ul RSB