18 -- 22 August notes

2014-08-18

Qubit for DGE libraries

KAPA library quant and Qubit results are off by an order of magnitude. ST recommended running Bioanalyzer before proceeding.

Talked to Illumnia reps about new technologies

NOTE - Bioan.nM were all diluted ~4 fold

Sample.Name	Qubit.nM	Bioan.nM	KAPA.nM
05-B-HS	65.19		0.2576
07-A-HS	273.9	17.22	1.155
07-B-CT	153.8	6.56	0.9051
08-D-CT	173.8	9.02	0.1989
11-C-CT	95.94		0.9481
15-A-CT	97.58	1.64	1.859
15-B-HS	277.2	4.92	4.145
20-C-CT	82.41		1.841
26-C-CT	53.71	11.89	0.8283
J1	51.66	4.92	0.4814
J2-A	47.56	0	0.5548
J3-A	35.67		0.6746
J4	116.4	4.51	0.6859
J1-P	31.16
J2-P	62.32	10.25

mean ratio Qubit to Kappa: 175

mean ratio Qubit to Kappa: 24.6

Comments from SHC

Bad news comes in threes. -80 freezer broke and up to 6C by the time we discovered it and moved samples.

Pooled samples using Qubit values for all to get 200nM for each sample

Total volume = 35.9 ul containing 2,730 nM for a final concentration of 76 nM/ul.

KAPA qPCR

Made 1e-3, 1e-4 and 1e-5 dilutions of pooled sample and ran KAPA qPCR.

Well	Sample.Name	Quantity	Cт	Dilution
A1	Std1	20		1
A2	Std1	20		1
A3	Std2	2		1
A4	Std2	2		1
A5	Std3	0.2	21.4	1
A6	Std3	0.2	10.59	1
A7	Std4	0.02	12.6	1
A8	Std4	0.02	14.33	1
A9	Std5	0.002	16.45	1
A10	Std5	0.002	16.51	1
A11	Std6	0.002	20.2	1
A12	Std6	0.002	20.45	1
B1	Pool-D1	971.9	8.949	0.001
B2	Pool-D1	264.5	9.822	0.001
B3	Pool-D1	362.6	9.61	0.001
B4	Pool-D2	0.00021	19.24	1e-04
B5	Pool-D2	0.00069	18.44	1e-04
B6	Pool-D2	0.00241	17.6	1e-04
B7	Pool-D3	0	32.15	1e-05
B8	Pool-D3	0	29.49	1e-05
B9	Pool-D3			1e-05

Standards 1 and 2 were ‘missing’ (pipetting error) and only consistent for Standards 5 and 6. Pipetting error?
Standards 5 and 6 okay, allowing rough quantification
Dilution 1 (10e-3) Ct ~ 9.5
Dilution 2 (10e-4) Ct ~ 18 but at edge of measurable range
Dilution 3 not measurable

Using only Dilution 1, median Quantity = 362, mean = 532

KAPA calculations of concentration 818 - 1,202 nM/ul

Qubit on the pooled sample: 22.2 ng/ul

Rough conversion to nM: 22.2 * 4.1 = 91 nM/ul

Baffling - this time the Qubit is an order of magnitude lower than KAPA…

Fixed Jekyll by upgrading to 13.10 following my own notes on stack overflow so back to updating website

James Waters visited lab. Went out to East Woods and showed him how to find Aphaenogaster - collected 2 queen-right colonies!

Writing…

Repeated KAPA qPCR.

Dilutions

Ran each dilution in triplicate with standards.

Beautiful results! r2 of standard curve = 0.99

Well	Sample.Name	Quantity	Cт	Dilution
A1	Std1	20	4.941	1
A2	Std2	2	8.357	1
A3	Std3	0.2	11.26	1
A4	Std4	0.02	14.76	1
A5	Std5	0.002	17.43	1
A6	Std6	2e-04	21.49	1
B1	Std1	20	4.691	1
B2	Std2	2	8.555	1
B3	Std3	0.2	11.21	1
B4	Std4	0.02	15.02	1
B5	Std5	0.002	18.36	1
B6	Std6	2e-04	21.57	1
C1	Std1	20	4.987	1
C2	Std2	2	8.632	1
C3	Std3	0.2	11.45	1
C4	Std4	0.02	14.61	1
C5	Std5	0.002	18.22	1
C6	Std6	2e-04	21.71	1
D1	Lib1	0.5856	9.99	0.001
D2	Lib1	0.4298	10.43	5e-04
D3	Lib1	0.1427	12.01	0.00025
E1	Lib1	0.4971	10.22	0.001
E2	Lib1	0.2283	11.34	5e-04
E3	Lib1	0.124	12.22	0.00025
F1	Lib1	0.4769	10.29	0.001
F2	Lib1	0.3325	10.8	5e-04
F3	Lib1	0.168	11.78	0.00025

Quantification of original library:

Sample.Name	KAPA.pM	KAPA.nM
Lib1	1,325	1.325

1.3 nM/ul / 4.1 = 0.32 ng/ul

Submitted qPCR amplified samples to Bioanalyzer for accurate estimation of library fragment size.