Latest revision as of 22:10, 26 October 2010

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

@@ Line 11: / Line 11: @@
-*'''Let's picked up colonys our transformant cells and prepare all to do miniprep.'''
+*'''Picked up colonies of transformant cells and set up all for miniprep.'''
-'''For this we used 30ml de LB liquid agar medium per falcon tube at 80μ/ml'''
+''' We used 30ml de LB medium per falcon tube with 35ng/ml'''
-'''Cloramphenicol concentration we left overnight culture to 37º '''
+''' of cloramphenicol  and cultured overnight at 37ºC '''
@@ Line 27: / Line 27: @@
 ==''Tuesday''==
-'''With a overnight inoculum  we did a miniprep.'''
-'''The miniprep was done with a Quiagen Kit.'''
+'''The miniprep was prepared using Quiagen Kit.'''
-'''Then we going to do a duble digest with EcoRI and PstI to check out our ligations.'''
+'''Then we did a double digestion with ''EcoR''I and ''Pst''I to check out the ligations.'''
@@ Line 59: / Line 58: @@
-==''Wensday''==
+==''Wednesday''==
-'''We run a gel to confirm our ligations as follow'''
+'''We ran a gel to confirm the ligations as follow:'''
 *'''PCR 1 Lig S with Psb1C3 Confirmed'''
@@ Line 71: / Line 70: @@
 [[Image:Conf3.gif|300px|left]]   [[Image:Con1.gif|300px|Right]]
-'''We prepare all to repeat ligation; PCR2 with Psb1C3 and PCR4 with Psb1C3'''
+'''We prepared all to repeat the ligation method: PCR2 with Psb1C3 and PCR4 with Psb1C3'''
@@ Line 77: / Line 76: @@
-'''We did ligations (Fast ligation one hour) Pcr 2 with Psb1C3 and PCR 4 with Psb1C3 then transformed DH5α'''
+'''We did ligations (The LIGator Kit Amresco) PCR2 with Psb1C3 and PCR 4 with Psb1C3, then transformed DH5α'''
-'''competent cells, and incubated at 37º overnight.'''
+'''competent cells, and incubated at 37ºC overnight.'''
-'''We ran a gel to test our biobricks for characterisation with the follow results'''
+'''We ran a gel to test  biobricks with the follow results:'''
 *'''I0260   Positive Plate 2010
@@ Line 94: / Line 93: @@
 ==''Friday''==
-'''We Checked the plates of previous day ligations and we have transformants for'''
+'''We checked the  previous day ligation plates and we get  colonies of'''
-'''PCR4 no for PCR2'''
+'''PCR4 and not of PCR2'''
 [[Image:Labo_259.JPG|400px]]
+'''Today we discussed the reasons why we have not been succesfull to confirm the PCR2'''
+'''maybe the ligation reaction is saturated'''
+[[Image:Ligation2.gif]]

Team:Mexico-UNAM-CINVESTAV/Notebook/Week Five

From 2010.igem.org