User:Cassandra M Barrett/Notebook/Open Chromatin/2015/09/05

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Ligase Cycling Reaction Attempt and Fragment Phosphorylation

Purpose: to attempt LCR with fragments amplified by Daniel (ATF2, and Gal4DB_mCh from KAH228) as well as my digested and blunted MV10.

Spent the morning sorting through old freezer boxes. Found necessary fragments and oligo bridges. Daniel already ran a gel to confirm all parts but I ran one again to confirm the ATF2 and Gal4DB_mCh parts. Gel was faint because it was old...but generally confirmed correct part size (if this LCR doesn't work I will reamplify both parts). Their concentrations are 84ng/uL and 65ng/uL respectively

Methods:

Begin by phosphorylating the parts. Unsure if phosphorylating the backbone will help or not. They did in the paper, but for blunt ligations the backbone is left unphosphorylated to prevent backbone reannealing to itself. I will try two reactions, one with phosphorylated bb and one with unphosphorylated bb.

90fmol of each part was phosphorylated as follows:

20 uL rxn for MV10 4uL MV10- 2uL 10X T4 PNK Buffer- 2uL 10mM ATP- 1uL T4 PNK 11uL Water-

20 uL rxn for ATF2 1uL ATF2- 2uL 10X T4 PNK Buffer- 2uL 10mM ATP- 1uL T4 PNK 14uL Water-

20uL rxn for Gal4DB_mCh 1uL Gal4DB_mCh- 2uL 10X T4 PNK Buffer- 2uL 10mM ATP- 1uL T4 PNK 14uL Water-

Incubate all three reactions at 37C for 30min. Inactivate at 65 for 20min


Set up LCR reaction components

Make a 300 nM working solution of oligo bridges (final volume = 100 μL) in a new tubes. 3 μL of 100μM oligo stock + 97 μL dH2O = 100 μL Use 1.0 μL of oligo working sln. per 10 μL LCR reaction

Fragments diluted as specified here : http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/04/06


Next step: Perform LCR, transform E.coli with 2.5uL of LCR reaction. Reaction stored in 4C fridge overnight