IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-6: Difference between revisions
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{{IGEM:/Harvard/2006/Cyanobacteria}} | |||
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<li>[[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-5 | Previous Entry]]</li> | |||
<li>[[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-6 | Current Entry]]</li> | |||
<li>[[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-7 | Next Entry]]</li> | |||
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==To-do== | ==To-do== | ||
# <s>Make frozen stocks of the KaiA+bkb and KaiB+bkb transformants that we grew up in liquid culture yesterday</s> | # <s>Make frozen stocks of the KaiA+bkb and KaiB+bkb transformants that we grew up in liquid culture yesterday</s> | ||
# Miniprep the above | # <s>Miniprep the above</s> | ||
# (Maybe) Do an X-P digest of the KaiA+bkb and KaiB+bkb plasmids (though we may want to wait and do this along with the other KaiA+bkb and KaiB+bkb ligation transformants) | # <s>(Maybe) Do an X-P digest of the KaiA+bkb and KaiB+bkb plasmids (though we may want to wait and do this along with the other KaiA+bkb and KaiB+bkb ligation transformants) '''(Will do later)'''</s> completed 8/7/06 | ||
# <s>Inoculate the KaiA+bkb and KaiB+bkb transformants from yesterday's ligation</s> | # <s>Inoculate the KaiA+bkb and KaiB+bkb transformants from yesterday's ligation</s> | ||
# <s>Add CIP to the E-S digest of LC vector</s> | # <s>Add CIP to the E-S digest of LC vector</s> | ||
# <s>Run the digests from yesterday on a gel (E-S digest of LC vector, X-P digest of J04450+bkb)</s> | # <s>Run the digests from yesterday on a gel (E-S digest of LC vector, X-P digest of J04450+bkb)</s> | ||
# Gel-extract and purify the LC vector and J04450 insert if they exist | # <s>Gel-extract</s> and purify the LC vector and J04450 insert if they exist '''(Will purify tomorrow, along with the high-copy backbone that the J04450 was inserted in)''' | ||
# (maybe) Perform a ligation and transformation of the J04450 insert into LC vector | # <s>(maybe) Perform a ligation and transformation of the J04450 insert into LC vector '''(Will do after we gel-purify them)'''</s> completed 8/7/06 | ||
# Do 12 minipreps for Mingming | # <s>Do 12 minipreps for Mingming</s> | ||
# <s> Check sequencing on the Genewiz website </s> (the orders have not been processed) | |||
==KaiA/B + bkb inoculation== | ==KaiA/B + bkb inoculation== | ||
We found 10 colonies on the KaiA+bkb plate and 5 colonies on the KaiB+bkb plate. We inoculated all 15 colonies in liquid culture (8 mL LB, 8 uL Amp) and placed them in the 37C shaker around 1830. | We found 10 colonies on the KaiA+bkb plate and 5 colonies on the KaiB+bkb plate. We inoculated all 15 colonies in liquid culture (8 mL LB, 8 uL Amp) and placed them in the 37C shaker around 1830. | ||
==Gel images of E-S digest of LC vector and X-P digest of J04450+bkb (high copy)== | |||
[[Image:2006-8-6 digests of BB J04450 and pSB4A3.jpg|thumb|left|Click for legend]] | |||
[[Image:2006-8-6 digests of BB J04450 and pSB4A3 dim.jpg|thumb|left|Click for legend]] | |||
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We extracted the high-copy vector (2kb) and J04450 insert (1kb) from lanes 2 and 4, and the low-copy vector (3.5 kb) from lanes 6 and 8. | |||
Latest revision as of 10:02, 10 August 2006
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To-do
Make frozen stocks of the KaiA+bkb and KaiB+bkb transformants that we grew up in liquid culture yesterdayMiniprep the above(Maybe) Do an X-P digest of the KaiA+bkb and KaiB+bkb plasmids (though we may want to wait and do this along with the other KaiA+bkb and KaiB+bkb ligation transformants) (Will do later)completed 8/7/06Inoculate the KaiA+bkb and KaiB+bkb transformants from yesterday's ligationAdd CIP to the E-S digest of LC vectorRun the digests from yesterday on a gel (E-S digest of LC vector, X-P digest of J04450+bkb)Gel-extractand purify the LC vector and J04450 insert if they exist (Will purify tomorrow, along with the high-copy backbone that the J04450 was inserted in)(maybe) Perform a ligation and transformation of the J04450 insert into LC vector (Will do after we gel-purify them)completed 8/7/06Do 12 minipreps for MingmingCheck sequencing on the Genewiz website(the orders have not been processed)
KaiA/B + bkb inoculation
We found 10 colonies on the KaiA+bkb plate and 5 colonies on the KaiB+bkb plate. We inoculated all 15 colonies in liquid culture (8 mL LB, 8 uL Amp) and placed them in the 37C shaker around 1830.
Gel images of E-S digest of LC vector and X-P digest of J04450+bkb (high copy)
We extracted the high-copy vector (2kb) and J04450 insert (1kb) from lanes 2 and 4, and the low-copy vector (3.5 kb) from lanes 6 and 8.
