IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/02: Difference between revisions

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**PCR purified digests
**PCR purified digests
***Concentrations: Pme (ranges from 20-36 ng/uL); Pal (ranges from 20 to 28 ng/uL)
***Concentrations: Pme (ranges from 20-36 ng/uL); Pal (ranges from 20 to 28 ng/uL)
**Ran backbone on gel and gel purified (Concentration: )
**Ran backbone on gel and gel purified (Concentration: 3.1 ng/uL)
 
*Ligations of Pme/Pal w/ V0120
{| border="1"
|+ ''Ligation Reactions''
!  !! Pme4 !! Pal3!! Pme5!! Pal1!! GFP(.5)!!
|-
! DNA Insert
| align="center" | 1.85 || align="center" | 1|| align="center" | 1|| align="center" | 4.86 after 1 in 10 dilution
|-
! T4 DNA ligase buffer (10x)
| align="center"  | 2 || align="center" | 2 || align="center" | 2|| align="center" | 2
|-
! diH<sub>2</sub>O
| align="center"  | 14.15 || align="center" | 15 || align="center" | 6.3|| align="center"  | --
|-
! T4 DNA ligase
| align="center"  | 1 || align="center" |  1 || align="center" |  1|| align="center" |  1
|-
! DNA Backbone
| align="center" | 1 (bbone 2) || align="center" |  1 (bbone 2)|| align="center" | 14|| align="center" | 9.64
|-
 
|}
 
**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)
* Check on transformations of amiRNA (5 transformations) and antisense/sense allergens (8 transformations)

Revision as of 12:05, 2 July 2010

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Team Allergy

  • Check on sequences for Bet
  • Digest Pme3 and Pal 2 introns/Ligate w/ V0120/Transform
    • Digestions
Digestion Reactions
PME 3 (4 digestions) Pal 2 (4 digestions) Backbone (B21 w/ YFP)
DNA 4 (~1000ng) 7 (~1000ng) 3 (~900 ng)
FD Buffer (10x) 2 2 2
diH2O 12 9 13
EcoRI 1 1 1
SpeI 1 1 1
    • 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
Ligation Reactions
Pme4 Pal3 Pme5 Pal1 GFP(.5)
DNA Insert 1.85 1 1 4.86 after 1 in 10 dilution
T4 DNA ligase buffer (10x) 2 2 2 2
diH2O 14.15 15 6.3 --
T4 DNA ligase 1 1 1 1
DNA Backbone 1 (bbone 2) 1 (bbone 2) 14 9.64
    • 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

Team Fence

pMT1002, pMT413 Digest

Digested with EcoR1 and HindIII

Lane 2 KB ladder

Lane 4 pMT1002

Lane 6 pMT413



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