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From 2010.igem.org

Week #5

04th October - 08 October 2010

Monday

• Let's picked up colonys our transformant cells and prepare all to do miniprep.

For this we used 30ml de LB liquid agar medium per falcon tube at 80μ/ml

Cloramphenicol concentration we left overnight culture to 37º

Tuesday

With a overnight inoculum we did a miniprep.

The miniprep was done with a Quiagen Kit.

Then we going to do a duble digest with EcoRI and PstI to check out our ligations.

DNA	5μl
Buffer NB2	1μl
EcoRI	0.5μl
PstI	0.5μl
H2O	2.7μl
BSA	0.3μl
Total	10μl

Wensday

We run a gel to confirm our ligations as follow

• PCR 1 Lig S with Psb1C3 Confirmed
• PCR 2 Lig S with Psb1C3 Unconfirmed
• PCR 3 Lig S with Psb1C3 Confirmed
• PCR 4 Lig C with Psb1C3 Unconfirmed

We prepare all to repeat ligation; PCR2 with Psb1C3 and PCR4 with Psb1C3

Thursday

We did ligations (Fast ligation one hour) Pcr 2 with Psb1C3 and PCR 4 with Psb1C3 then transformed DH5α

competent cells, and incubated at 37º overnight.

We ran a gel to test our biobricks for characterisation with the follow results

• I0260 Positive Plate 2010
• E0240 Positive Plate 2010
• R010 Positive Plate 2010
• J13002 Negative Plate 2007