IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/26

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Team Fence
PFX polymerase PCR


 * 1μL 10mM MgSO4
 * 2μL 10X pfx amplification buffer
 * 2μL 10X PCRx enhancer solution
 * 1μL F primer
 * 1μL R primer
 * 0.5μL pfx polymerase
 * 2μL DNTP

9.5μL total

DNA: 21ng/μL
 * 50 to 200μL DNA per reaction
 * 5 reactions
 * 4μL per reaction = 20μL sample total (MM)

9.5μL + 4μL = 13.5μL therefore 6.5μL H20 per reaction

MM


 * 5μL 10nM MgSO4
 * 10μL 10X pfx amplification buffer
 * 10μL 10X PCRx enhancer solution
 * 5μL F primer
 * 5μL R primer
 * 2.5μL pfx polymerase
 * 10μL DNTP
 * 20μL genomic DNA
 * 32.5μL H20

total volume:100μL

Transformations

 * Thawed 2 turbo cells on ice to thaw
 * Resuspended EcR, RXRHm, RXRLc tubes from Mr.Gene into 50μL DH2O (5μg into 50μL)
 * Pippetted .5μL plasmid from each of the 5 synthesis products (EcR, RXRHm, RXRLc, ACC, and ACT2LacOpt) into a labled eppendorf containing 20μL of turbo cells each
 * Pippetted 2μL of the relavent ligation reaction from Friday (Strep.Barnase 2 ligation, Strep.Barnase 1, Gal4 DBD lig) into eppendorfs containing 20μL turbo cells (barn1strep, barn2strep and Gal4DBD)
 * Chilled on ice for 15 mins
 * Put in 42°C heat bath for 30 secs
 * Put on ice for 2 mins
 * Added 200μL SOC medium (to equal 10x the amount of cells)
 * Set to shake at 37°C for 30 mins (longer for Act2pt because kan selective cultures need more time to become established before facing the harsher Kan selection medium
 * Streaked 20μL on LB+amp plates
 * Put in incubator to shake overnight
 * After 45 mins to 1.5 hours, streaked Act2LacOpt on Kan+LB plate, put in incubator to shake overnight.

Making LB+Kan Plates
15μL Kanamycin 30μL DH2O to ease streaking Streaked this on LB agar plates with glass beads, and then turned the plate upside down and let dry until it was ready for bacterial sequencing, and then those same beads were reused.
 * made 4 LB+Kanamycin plates

Transformation of pLAS and Terminator
Resuspended the following from the registry with 10μL DH2O p(LAS)TetO, on pSB2K3 (kan res.), from plate 2, sell 11N Terminator, on pSB1AK3 (kan and amp res.), from plate 1, well 4H

Procedures
Last week, we finished ligation together Sense and Intron parts of the hpRNA and transformed them into E. coli. This week, we will extract the plasmids, add in the antisense part, and transform.

Procedures today...
 * 1) Culture colonies (done over the weekend)
 * 2) Miniprep of V0120 sense + PDK, PAL intron parts
 * 3) Diagnostic Digest

We also need to check which parts of the sequence on the team page corresponds to the sense/antisense parts of the gene. There may have been mistakes in the past.

Results
Culture We cultured 38 colonies. All grew but one LTP+PAL and Ger+Pal grew. We labeled the cultures 1- 38 for simplicity sake.

1-6: LTP + PDk 7-16: Miniprep

Here are the concentrations of the miniprepped plasmid.

Diagnostic Digest

We digested the vectors with Spe and Pst1 and ligate with antisense parts (already digested and gel purified). We ran them on the E Gel too long so the lanes do not show anything conclusive, other than that there is digested DNA in the bottom half. Result:

Sequence review clarified which parts of the sequences on the team page corresponded to sense/antisense.

Team Flavor

 * Digested miniprepped Mira/Brazz+StrepII+STOP constructs with Not1/Spe1 for ligation confirmation: failed
 * Ran PCR product of Valencia Orange genome to confirm: failed

Digestion Reactions Mira N: 888ng Mira C: 888ng Brazz N: 993ng Brazz C: 1032ng STOP:   781ng x 2 (max DNA available)
 * Digested Mira/Brazz+StrepII constructs EcoRI/SpeI, STOP+V0120 EcoRI/XbaI


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