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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

Friday

We Checked the plates of previous day ligations and we have transformants for

PCR4 no for PCR2

Today we discussed the reasons why we have not been succesful to confirm the PCR2

Ligation maybe the reaction is saturated