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

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(Team Allergy)
(Team Allergy)
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==Team Allergy==
==Team Allergy==
-
*minipreps of v0120sense+pdk/pal intron parts
+
===Procedures===
-
*Digest vectors w/ Spe and Pst1 and ligate with antisense parts (already digested and gel purified)
+
 
-
**Result: [[Image:2-assembly_digest.jpg|50px]]
+
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.
-
*Sequence review: clarified which parts of the sequences on the team page corresponded to sense/antisense.
+
 
 +
Procedures today...
 +
#Culture colonies (done over the weekend)
 +
#Miniprep of V0120 sense + PDK, PAL intron parts
 +
#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.
 +
 
 +
{| border="1"
 +
|+ '''Sample Number'''
 +
!  !! 1 !! 2 !! 3 !! 4 !! 5 !! 6 !! 7 !! 8 !! 9 !! 10
 +
|-
 +
! LTP+PDK
 +
| align="center" | 233.9 || align="center" | 507.6 || align="center" | 498.7 || align="center" | 510.0 || align="center" | 485.1 || align="center" | 588.3 || align="center" |  || align="center" |  || align="center" |  || align="center" |
 +
|-
 +
! LTP+PAL
 +
| align="center" | 471.8 || align="center" | 586.7 || align="center" |500.2 || align="center" | 449.4 || align="center" | 492.7 || align="center" | 430.0 || align="center" | 521.8 || align="center" | 605.8 || align="center" | 499.1 || align="center" | 299.9
 +
|-
 +
!Bet v1+PDK
 +
| align="center" | 430.1|| align="center" | 519.3 || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |
 +
|-
 +
! Bet v1+PAL
 +
| align="center" | 432.3 || align="center" | 300.6 || align="center" | 458.7 || align="center" | 213.8 || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |
 +
|-
 +
! Bet v2+PDK
 +
| align="center" | 270.3 || align="center" | 223.3 || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |
 +
|-
 +
! Bet v2+PAL
 +
| align="center" | 270.3 || align="center" | 223.3 || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |
 +
|-
 +
! Bet v2+PDK
 +
| align="center" | 270.3 || align="center" | 223.3 || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |
 +
|-
 +
! Ger3+PDK
 +
| align="center" | 440.4 || align="center" | 205.2 || align="center" | 342.7 || align="center" | 290.2 || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |
 +
|-
 +
! Ger3+PAL
 +
| align="center" | 335.4 || align="center" | 297.8 || align="center" | 474.3 || align="center" | 396.1 || align="center" | 354.7 || align="center" | 366.2 || align="center" |  || align="center" |  || align="center" |  || align="center" |
 +
|-
 +
|}
 +
 
 +
'''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: [[Image:2-assembly_digest.jpg|right|200px]]
 +
 
 +
'''Ligation of Antisense Part'''
 +
 
 +
While another Diagnostic Digest was being set up, we went ahead and ligated all 38 parts with antisense.
 +
 
 +
'''Sequence review''' clarified which parts of the sequences on the team page corresponded to sense/antisense.
==Team Flavor==
==Team Flavor==

Revision as of 10:34, 30 July 2010

iGEM iGarden Main project page
Previous entry      Next entry

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

Arabidopsis PCR

Image: 726arapcregel.jpg-px240‎

Team Allergy

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.

Sample Number
1 2 3 4 5 6 7 8 9 10
LTP+PDK 233.9 507.6 498.7 510.0 485.1 588.3
LTP+PAL 471.8 586.7 500.2 449.4 492.7 430.0 521.8 605.8 499.1 299.9
Bet v1+PDK 430.1 519.3
Bet v1+PAL 432.3 300.6 458.7 213.8
Bet v2+PDK 270.3 223.3
Bet v2+PAL 270.3 223.3
Bet v2+PDK 270.3 223.3
Ger3+PDK 440.4 205.2 342.7 290.2
Ger3+PAL 335.4 297.8 474.3 396.1 354.7 366.2

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:

Ligation of Antisense Part

While another Diagnostic Digest was being set up, we went ahead and ligated all 38 parts with antisense.

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

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



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