IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-30: Difference between revisions
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==Ligation Protocol I used== | |||
Go to our Protocols section, extensive writeup. | |||
==Ligation #6 Plates== | ==Ligation #6 Plates== | ||
Thousands of colonies! Excuse the blurriness, used my cameraphone :( | Thousands of colonies! Excuse the blurriness, used my cameraphone :( | ||
[[Image: molar2_830.jpg|thumb|Molar Ratio, 2uL ligation product]] | [[Image: molar2_830.jpg|thumb|left|Molar Ratio, 2uL ligation product]] | ||
[[Image: molar19_830.jpg|thumb|Molar Ratio, 19uL ligation product]] | [[Image: molar19_830.jpg|thumb|left|Molar Ratio, 19uL ligation product]] | ||
[[Image: volume2_830.jpg|thumb|Volume Ratio, 2uL ligation product]] | [[Image: volume2_830.jpg|thumb|left|Volume Ratio, 2uL ligation product]] | ||
[[Image: volume19_830.jpg|thumb|Volume Ratio, 19uL ligation product]]<br | [[Image: volume19_830.jpg|thumb|left|Volume Ratio, 19uL ligation product]] | ||
<br style="clear:both;"/> | |||
'''Note that for 2uL we got a lot more colonies than for 19uL. Means that the ligation mixture is toxic to the cells; do not use 19uL anymore for chemically competent cells!''' | '''Note that for 2uL we got a lot more colonies than for 19uL. Means that the ligation mixture is toxic to the cells; do not use 19uL anymore for chemically competent cells!''' | ||
==Finally== | ==Finally== | ||
Did a colony PCR of 36 colonies + positive control, streaking each on part of a KAN plate. 8uL PCR supermix, 1uL VF2 (2nM), 1uL VR (2nM). | |||
It looks like we've finally succeeded in ligating J04500\SP + KaiC\XP! | It looks like we've finally succeeded in ligating J04500\SP + KaiC\XP! | ||
| Line 16: | Line 25: | ||
[[Image:cyano_2006-8-30_J04500 and KaiC_1_2.jpg|thumb|left|click for caption]] | [[Image:cyano_2006-8-30_J04500 and KaiC_1_2.jpg|thumb|left|click for caption]] | ||
[[Image:cyano_2006-8-30_J04500 and KaiC_3.jpg|thumb|left|click for caption]] | [[Image:cyano_2006-8-30_J04500 and KaiC_3.jpg|thumb|left|click for caption]] | ||
<br style="clear:both;"/> | |||
Succesful ligations: V2 #4, V19 #1, #4, #5, #6, #7, M2 #7, M19 #2, #4, #8 | |||
==Sequencing results for KaiB== | |||
... Don't make sense. Doing some AlignX work, shows that KaiB WITH biobricks prefix and suffix is in front of the J04500 promoter+RBS. Ideas? | |||
Latest revision as of 22:18, 30 August 2006
Ligation Protocol I used
Go to our Protocols section, extensive writeup.
Ligation #6 Plates
Thousands of colonies! Excuse the blurriness, used my cameraphone :(
Note that for 2uL we got a lot more colonies than for 19uL. Means that the ligation mixture is toxic to the cells; do not use 19uL anymore for chemically competent cells!
Finally
Did a colony PCR of 36 colonies + positive control, streaking each on part of a KAN plate. 8uL PCR supermix, 1uL VF2 (2nM), 1uL VR (2nM).
It looks like we've finally succeeded in ligating J04500\SP + KaiC\XP!
Succesful ligations: V2 #4, V19 #1, #4, #5, #6, #7, M2 #7, M19 #2, #4, #8
Sequencing results for KaiB
... Don't make sense. Doing some AlignX work, shows that KaiB WITH biobricks prefix and suffix is in front of the J04500 promoter+RBS. Ideas?
