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From 2010.igem.org

Week #5

04th October - 08 October 2010

Monday

• Picked up colonies of transformant cells and set up all for miniprep.

We used 30ml de LB medium per falcon tube with 35ng/ml

of cloramphenicol and cultured overnight at 37ºC

Tuesday

The miniprep was prepared using Quiagen Kit.

Then we did a double digestion with EcoRI and PstI to check out the ligations.

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

Wednesday

We ran a gel to confirm the 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 prepared all to repeat the ligation method: PCR2 with Psb1C3 and PCR4 with Psb1C3

Thursday

We did ligations (The LIGator Kit Amresco) PCR2 with Psb1C3 and PCR 4 with Psb1C3, then transformed DH5α

competent cells, and incubated at 37ºC overnight.

We ran a gel to test biobricks with the follow results:

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

Friday

We checked the previous day ligation plates and we get colonies of

PCR4 and not of PCR2

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

maybe the ligation reaction is saturated