IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/02

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Team Allergy

 * Check on sequences for Bet
 * Digest Pme3 and Pal 2 introns/Ligate w/ V0120/Transform
 * Digestions


 * PCR purified digests
 * Concentrations: Pme (ranges from 20-36 ng/uL); Pal (ranges from 20 to 28 ng/uL)
 * Ran backbone on gel and gel purified (Concentration: 3.1 ng/uL)


 * Ligations of Pme/Pal w/ V0120


 * bbone 2 was potentially contaminated at a higher concentration 96.1 ng/uL


 * Check on transformations of amiRNA (5 transformations) and antisense/sense allergens (8 transformations)
 * There were colonies from the amiRNA transformations
 * We grew up 24 colonies (3 for each GFP, GFP .5, LTP, LTP .5, BET) ~ 10 am to be ready ~3 pm for minipreps)
 * The other 8 transformations did not show any colonies--we put it back in to see if we would get colonies (only 100 uL instead of 200 uL was plated)
 * To do: Grow up colonies to miniprep, run on gel, sequence


 * pHannibal and pKannibal arrived today (hannibal is amp resistant and kannibal is kan resistant)
 * We transformed these plasmids

pMT1002, pMT413 Digest
Digested with EcoR1 and HindIII

Lane 2 KB ladder

Lane 4 pMT1002

Lane 6 pMT413



Barnase PCR
Standard PCR measurements were used except that a new 50X dNTP mix was used in place of the 5X buffer used in all previous PCR reactions. The amount of dH20 was adjusted for the new quantity of dNTP mix accordingly.




 * lane 1: ladder
 * lanes 2-5: PMT413 temperature gradient
 * lanes 6-9 PMT1002 temperature gradient

As this was our 3rd or 4th or 5th unsuccessful Barnase PCR Oliver checked the primers to discover a glitch in the forward primer, it seemed to have some extra nucleotides inserted and also had some complementarity to the adjacent gene on the plasmid barstar. New primers will be ordered.

Ligation and Cloning
Ligation performed in LacIN as colonies were seen for Barstar and NLS
 * 3 times as much insert as vector per reaction, length of insert taken into consideration.
 * LacIN: 1.1 kb = 51 ng

Ligation Recipe:
 * 10μL insert + backbone
 * 10μL ligation buffer (vortexed thoroughly)
 * 1μL ligase, mix gently

let stand for 15 minutes at room temperature, then put on ice use for 4μL of the product for transformation and freeze the rest

LacIN
 * 3μL B21 vector
 * 7μL LacIN insert

as the LacIN DNA found did not have the concentration or quantity labeled the DNA was vacufuged and resuspended in 12.6μL of dH20 after nanodrop values indicated the total quantity of DNA was ≈90ng and our desired quantity was 51ng

Transformation from ligation
 * thawed 4 chemically competent BL21 cell tubes on ice
 * added 5μL of each ligase rxn to each incubated on ice for 30 minutes
 * with the following exception: all of the remaining NLS ligase reaction from the previous day was added
 * heat shock at 42°C for 30 seconds
 * put on ice for 2 minutes
 * added 170μL SOC medium to each tube set to shake at 37°C for 30 minutes
 * pippetted each tube onto its own LB+amp plate, streak with beads
 * incubate at 37°C overnight

Colonies from yesterday's ligation
no colonies on B21 negative control

colonies!

no colonies ligation to be performed again

a small number of colonies

NosT + stop and v0120
We ran an xba/pstI fast enzyme digest on our miniprepped NosT + stop and v0120 plasmid. The gel was consistent with a successful ligation. (See lanes 7-10 in gel image below).



Valencene
After running a reverse transcriptase reaction on our RNA extraction, we ran PCR on the cDNA with Valencene specific primers. The gel did not show the proper fragment length of the Valencene gene. Instead, it appears primer dimers formed again. (See lanes 2-5)