Lab Notebook - Joseph

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B277 PCR of basic parts from construction file

6/23/09 /wiki/Construction_Files#PCR_Construction_of_.7B.3CespP.28beta.29.3E.7D

protocol used: Template:SBB-Protocols PCR2

all 2K55 cycling program, except B09ig328, run at 4K55

PCR Construction of {<espP(beta)>} PCR Construction of {<upaG_short>} PCR Construction of {<CPG_L2>} PCR Construction of {<CPG_L6>} PCR Construction of {<TshA>} PCR Construction of {<eCPX>} PCR Construction of {<ehaB>} Basic PCR construction of B09ig324 {<SOD>} Basic PCR construction of B09ig326 {<Tir-M>} Basic PCR construction of B09ig328 {<cel6A!}


PCR of basic parts from construction file (cont'd)

Cont'd PCR products (x10)from yesterday were zymocleaned, analyzed on gel, digested with Eco/Bam as per protocol, gel purified, Zymo column purif, and frozen. In addition, 3 ul of vector 9523-1439 obtained from V, was Eco/Bam digested for general use. Analytical gel showed all 10 PCR products in expected size range.


Elicia and Joey ligated the 10 digested,gp'd PCR products using 9523-1439 parent vector and transformed them into___ competent cells, plating on LB/?

Streptavidin assay development


Transformation of DH10B with pBca9145-9494 for use as positive control in streptavidin assay= Done with protocol Lane modified Did both +/- plasmid. Used AMP plates from rm327, good XF. Using AMP plates from B144, lawns grew on both plates, suggesting no AMP on plates (sleeve of AMP plates tossed)


Picked colonies from pBca9145-9494 (putative +)plate into 3x 4ml cultures for SA assay Picked colonies from pBca9495-1393(putative -) plate into 3x 4ml cultures for SA assay


Ran above culture +/- arab using 35λ cells and 5λ SA at 40μg/mL using 384 well format (protocol: Results: high fluor in all wells, imaged upside down (USD) and right side up (RSU) with USD showing more plastic artifact brightness but also clearer pellet with spun cells. After transfer of sup to new wells(difficult) and adding PBS to cells, most fluor went with sups but still some in cells, tho inconsistent. This procedure does not seem accurate, nor feasible and a larger scale. Plan to try 96 conical , to allow packed pellet and more cells.


O/N DH10B cultures with pBca9145-9494 and pBca9495-1393 were diluted 1:100 (to max cell# at 5hrs induction) and 9494 cells only were induced with 100μg/mL arabinose for possible use this PM with new SA.


Tested ELESA protocol - Used ARA-induced (100 μg/ml for 5 hrs)pBca9145-9494 and uninduced pBca9495-1393 cells diluted 1:100 from O/N cultures. 96 well flat bottom plates used, one well each , no cell control. We may want to consider re-inducing culture at say, 3 hrs as cell # increases.


Did a mini-prep with Elicia, but used Zymo columns by mistake. We started a re-do.


Did mini-prep of ig280, ig282, and ig283.


I ran an analytical gel of mini-prepped plasmids ig280, ig282, and ig283.


Did mini-prep of 9523-M10053, M10037, M10022, 9523-M10025, M10087, M10046, M10018, and M10089.

Restarting Reverse Gateway


Did transformation of pBca1256-k112126 and pBca1256-mv45 to TG1 cells. Plated on Spec.


Did an EcoRI/BamHI analytical digest mapping of mv45, k112126, and 9145-9494. Then proceeded with an EcoRI/BamHI digest of vector plasmids mv45 and k112126. Patrick completed the gel purification step.


Did colony PCR of mv45 and k112126.


Did a mini-prep of mv45. Couldn't proceed with the mini-prep of k112126 because they were RFP colonies.


Did a mini-prep of inoculated colonies (that Pat did) of k112126 (1) and (2). Then proceeded to do a digestion of mini-prepped mv45 and k112126 (1) and (2). Ran a 10-μL-digest gel of each, and later co-gel purified appropriate bands of mv45 and k112126 (2), and finally, did a bench-top ligation, all according to Madhvi Venkatesh's "Protocol for Assemblies by Hand." Transformed the ligation mixture to Lefty Cells and plated on Amp.


Nothing grew from yesterday's plating. Started with the mini-prepped plasmids mv45 and k112126 (2) and ran three digests: two according to Madhvi Venkatesh's "Protocol for Assemblies by Hand" for mv45 and k112126 and one digest mapping for k112126 cut only with BglII (not even EcoRI). Ran a gel for the three digests. Pat cut out the appropriate bands for mv45 and k112126 and gave them to me to continue with the gel-purification and ligation. Transformed ligation mixture to Lefty cells and plated on Amp.


One colony grew from yesterday's plating; picked and placed in 5 mL of Amp LB. Did a ligation reaction using yesterday's eluted material. Transformed ligation mixture into lefty cells. Although the cell mixture is plated on Amp, I rescued them anyway to see if that had any effect on yesterday's poor colony growth.


Ran a gel for Patrick's ATT (1), (2), (3), and RBS (1), (2), and "Cellulose;" the bands weren't as expected. Did a digestion of ATT (1) and RBS (2) for Patrick. The cell mixture plated on 8/8/09 grew successfully; picked three colonies and placed them in Amp.


Did a mini-prep and EcoRI/BamHI digest of mv45-k112126 (1), (2), and (3); ran a gel, and no bands showed up. Picked another set of three colonies for tomorrow. Did a small frag Zymo cleanup of Pat's two ATTR2 parts and ligated one to the "9145" vector and the other to the "4599" vector; transformed each ligation to Righty cells and plated on Amp.


Did a mini-prep of the three mv45-k112126 colonies picked yesterday; ran a gel that showed faint signals. Sent one of them off for sequencing. The plated cells from yesterday grew successfully; picked two from each and placed them in Amp.


Did a mini-prep of two ATTR2-4599 and one ATTR2-9145 cells.