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From 2013.igem.org

To design the Linker Variation Constructs, we amplified the DNA fragments LV1a, LV1b, LV2b, LV3a, LV5b and LV6 by PCR with the following protocol. (Primer are listed on our primer-page)

What	µl
primer_fw (see link above)	2
primer_rv (see link above)	2
Brevibacillus parabrevis	1
Phusion Flash 2x Master Mix	10
ddH20	5

PCR-Program LV1a, LV1b, LV2b, LV5b amplification

Cycles	Temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:02
	59,4	0:05
	72	0:28
1	72	10:00
1	10	inf

PCR-Program LV3a, LV6a amplification

Cycles	Temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:02
	59,4	0:05
	72	0:44
1	72	10:00
1	10	inf

Contents 1 Results 2 Gel extraction 3 Results 4 Amplification of Backbone and indC 5 Results

Results

We were able to amplify all of our constructs, each of them run on the expected size.

Gel extraction

The amplified constructs LV14,LV1b,LV2b,LV3a,LV5b,LV6a were sliced out of the gel and a gel extraction performed. Afterwards, the concentration was measured with a nanodrop.

Results

Construct	Concentration in ng/µl	A280/A260
LV1a	41,6	1,89
LV1b	69,4	1,86
LV2b	74,4	1,97
LV3a	72,9	1,84
LV5b	56,7	1,94
LV6a	50,7	2,05

Amplification of Backbone and indC

As we want to usethe strategy we established to detect a successful transformation by the expression of indigoidine, we amplified not only the backbone, but also the indigoidine constructs. They were amplified with both, a gibson-overhang for the short-linker-constructs and the long-linker-constructs.

PCR-Program backbone short- and long-linker amplification

Cycles	Temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:02
	66,2	0:05
	72	0:50
1	72	10:00
1	10	inf

PCR-Program indigoidine short- and long-linker amplification

Cycles	Temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:02
	56,1	0:05
	72	1:20
1	72	10:00
1	10	inf

Results

As we were able to amplify all af the constructs, we quantified the gained concentrations for gibson assembly.

20130920 amplificationbb+ind.png Constructs of bb and indC after PCR.

20130919 20130922 quantificationbb+ind.png Concentrations of bb- and indC-constructs.

Construct	Concentration ind ng/µl
BBs	10
BBl	40
Inds	45
Indl	45