"

From 2013.igem.org

Contents 1 Results of liquid cultures 2 Minipreps using the Qiagen kit 3 Glycerol stocks 4 NanoDrop from miniprep 5 Digestion 6 In gel

Results of liquid cultures

Some of our liquid cultures didn't work; the cells died in the antibiotic. Mildly frustrating. The failed cultures were:

• OmpR (BBa_K098011)
• Lambda inverter system (BBa_Q04510)

Minipreps using the Qiagen kit

We miniprepped:

• CcaR

• Promoter, RBS x 3 replicates

• CcaS

• B0034

• Magenta

• OmpR promoter

• OmpR

• YF1

• Lambda inverta

• Fix J

• Yellow

• Fix J promoter

Glycerol stocks

We made glycerol stocks of what is above, then stored them at -80^oC

For future reference, to get glycerol stocks back as plates, take a loop and streak on gel. Has to be taken from frozen glycerol stocks, which are immediately returned to the freezer.

We then made a gel for digests using ethidium bromide.

NanoDrop from miniprep

Part	Concentration (ng/ul)
CcaR	324.6
CcaS	264.4
Yellow	64.3
Magenta	102.7
RBS	48.9
Promoter, RBS, #1	21.5
Promoter, RBS, #2	22.6
Promoter, RBS, #3	22.0
Fix L	31.9
Fix J	35.4
YF1	27.1
OmpF	28.2

No SureClean was needed.

Digestion

We then cut the genes out of the plasmid using Xbal and Pstl. Prepare to run on a gel.

Each eppendorf contains:

• 12ul purite water

• 2ul 10X FastDigest Buffer w.Green

• 0.5ul Xbal

• 0.5ul Pstl

• 5ul DNA

In gel

Lane	Content
1	Ladder (1kb Gene Ruler)
2	Promoter, RBS, #1
3	B0034
4	CcaS
5	Fix J promoter (a.k.a Fix L)
6	Yellow
7	Promoter, RBS, #2
8	Fix J
9	OmpR promoter (a.k.a OmpF)
10	Promoter, RBS, #3
11	Magenta
12	CcaR
13	YF1
14	Ladder (1kb Gene Ruler)

The gel shows that some of our digestions appear to have worked (our yellow pigment in lane 6 has a clear band at about ~700bp, where would expect) but others appear not to have worked, or have bands that don't match our expected genes.

We also ran a seperate gel of:

• cph8 #3

• cph8 #4

• cph8 #5

• cph8 #6

• cph8 #7

Cut with Xbal and Pstl.

The lanes were run in the order above, flanked by a 1kb Gene ruler.