IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/27

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(Mira/Brazz+StrepII+STOP confirmation digest)
(Team Allergy)
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==Team Allergy==
==Team Allergy==
-
* Diagnostic Digest (EcoR1 & Pst1)
+
===Procedures===
 +
Yesterday, we tried to run a diagnostic digest of the Sense + Intron hpRNA. The gel did not work properly, so we are redoing the gel today. We'll also go ahead and ligate antisense parts into all 38 minipreps that we HOPE contains correct Sense + Intron. Once the gel finishes running, we can go back and dispose of the ligations today that do not contain the correct S+I parts.
 +
 +
Today, we are also going to redo the ligation of Sense + Intron for PDK colonies because the gel that we are redoing today suggests that no PDK worked from last week's ligation.
 +
 +
Procedures
 +
#Diagnostic Digest
 +
#Diagnostic Gel
 +
##Diagnostic Gel of PDK + Sense
 +
#Ligation of Antisense Part into 38 minipreps
 +
 +
Redoing ligations for Sense + PDK
 +
#Digest of pLTP, pBet v1, pGer with S, P
 +
#PCR purification
 +
#Ligation of Sense+PDK
 +
#Transform and Plate
 +
 +
===Results===
 +
 +
'''Diagnostic Digest'''
 +
 +
38 digests of the sense+intron miniprepped plasmid DNA. The digest was done with Slow Digest Eco and Pst
 +
 +
'''Diagnostic Gel'''
 +
 +
From the digest, we can see that two LTPS+Pal worked (9,11), 3 Ger3S+Pal worked (36-38), 4 Bet2S+Pal Worked (25-28)
 +
 +
Lanes are:
1-6: LTP + PDK  (3200 & 1200);
1-6: LTP + PDK  (3200 & 1200);
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Image: Ladder (every 6)
Image: Ladder (every 6)
-
[[Image: Ddigestrun2.jpg|240px]]
+
[[Image: Ddigestrun2.jpg|350px]] [[Image: Ddigestrun3.jpg|350px]]
-
[[Image: Ddigestrun3.jpg|240px]]
+
Rerun of the lanes that were ligations of PDK + sense because they did not show on the gel above. We feel that there might be a chance that if the Sense+PDK assembly did not work, the 300bp evidence ran off the first gel. This second gel shows that most of the PDK+sense did NOT work. Only three worked -- minipreps 16, 30, and 31. 
-
Rerun: [[Image:2-assembly-diagnost-L.jpg|50px]] [[Image:2-assembly-diagnost-R.jpg|50px]]
+
[[Image:2-assembly-diagnost-L.jpg|350px]] [[Image:2-assembly-diagnost-R.jpg|350px]]
-
* Two LTPS+Pal worked (9,11), 3 Ger3S+Pal worked (36-38), 4 Bet2S+Pal Worked (25-28)
 
-
**Digested these parts with spe and pst1 (slow digest)
 
-
''Antisense-Intron Backbone Digestion''
+
'''Ligated with antisense parts and transformed'''
 +
 
 +
Digested antisense parts with X, P and pSense+Intron with S, P. Digestion ran for 2 hours, with an additional 20 minutes to de-phosphatize the backbone. Ligations were done for colonies that showed correct Sense+Intron ligations.
{| border="1"
{| border="1"
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|}
|}
-
(Let it go for 2 hours and then phosphatized backbone (20 min)
+
'''Transform and Plate'''
 +
 
 +
The standard Turbo Cell transformation protocol was followed.
 +
 
 +
'''Redo Ligations for PDK+ Sense'''
 +
 
 +
Since the gel showed that most of them did not work.
 +
 
 +
Digestion of pLTP, pBet v1, and pGer 3 with S, P to prepare them as a back bone. Phosphatase was used. Did not digest pBet v2 because there was not enough.
 +
 
 +
PCR purify the digested backbones containing Sense.
 +
 
 +
''Nanodrop''
 +
 
 +
pLTP - 24.0
 +
pBet - 17.2
 +
pGer - 42.8
-
*Ligated with antisense parts and transformed
+
Ligate predigested PDK digested with X, P and ligate into purified backbone. Transform and Plate (6? plates total)
==Team Flavor==
==Team Flavor==

Revision as of 13:30, 30 July 2010

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Team Allergy

Procedures

Yesterday, we tried to run a diagnostic digest of the Sense + Intron hpRNA. The gel did not work properly, so we are redoing the gel today. We'll also go ahead and ligate antisense parts into all 38 minipreps that we HOPE contains correct Sense + Intron. Once the gel finishes running, we can go back and dispose of the ligations today that do not contain the correct S+I parts.

Today, we are also going to redo the ligation of Sense + Intron for PDK colonies because the gel that we are redoing today suggests that no PDK worked from last week's ligation.

Procedures

  1. Diagnostic Digest
  2. Diagnostic Gel
    1. Diagnostic Gel of PDK + Sense
  3. Ligation of Antisense Part into 38 minipreps

Redoing ligations for Sense + PDK

  1. Digest of pLTP, pBet v1, pGer with S, P
  2. PCR purification
  3. Ligation of Sense+PDK
  4. Transform and Plate

Results

Diagnostic Digest

38 digests of the sense+intron miniprepped plasmid DNA. The digest was done with Slow Digest Eco and Pst

Diagnostic Gel

From the digest, we can see that two LTPS+Pal worked (9,11), 3 Ger3S+Pal worked (36-38), 4 Bet2S+Pal Worked (25-28)

Lanes are: 1-6: LTP + PDK (3200 & 1200);

7-16: LTP + PAL (3200 & 540);

17,18: Bet1 + PDK (3200 & 1200);

19-22: Bet1 + PAL (3200 & 540);

23,24: Bet2+PDK (3200 & 1200);

25-28: Bet2 + PAL (3200 & 540);

29-32: Ger3 + PDK (3200 & 1200);

33-38: Ger3 + PAL (3200 & 540);

Image: Ladder (every 6)

Rerun of the lanes that were ligations of PDK + sense because they did not show on the gel above. We feel that there might be a chance that if the Sense+PDK assembly did not work, the 300bp evidence ran off the first gel. This second gel shows that most of the PDK+sense did NOT work. Only three worked -- minipreps 16, 30, and 31.


Ligated with antisense parts and transformed

Digested antisense parts with X, P and pSense+Intron with S, P. Digestion ran for 2 hours, with an additional 20 minutes to de-phosphatize the backbone. Ligations were done for colonies that showed correct Sense+Intron ligations.

1-39
DNA ~1.5 ug
10x BSA 3
diH2O Fill to 30
Spe 1.5
Pst1 1.5
Neb Buffer 2 (10x) 3

Transform and Plate

The standard Turbo Cell transformation protocol was followed.

Redo Ligations for PDK+ Sense

Since the gel showed that most of them did not work.

Digestion of pLTP, pBet v1, and pGer 3 with S, P to prepare them as a back bone. Phosphatase was used. Did not digest pBet v2 because there was not enough.

PCR purify the digested backbones containing Sense.

Nanodrop

pLTP - 24.0 pBet - 17.2 pGer - 42.8

Ligate predigested PDK digested with X, P and ligate into purified backbone. Transform and Plate (6? plates total)

Team Flavor

Ligation of Mira/Brazz + StrepII to STOP + V0120

  • Insert to Backbone ration of 3:1
   Miraculin: 34ng
   Brazzein:  11ng
   Backbone:  50ng
  • Ligations were transformed, plated onto LB+AMP and left overnight @ 37°C

PCR of Valencene using Pfx Polymerase

  • Pfx polymerase was rumoured to be more accurate at PCR from genomic DNA
   Specs:
    DNA 1-1: 1.8 ng/μL
    DNA 1-2: 1.1 ng/μL
    DNA 2-1: 2.6 ng/μL
    DNA 2-2: 2.2 ng/μL

Mira/Brazz+StrepII+STOP confirmation digest

   Digested DNA:
    MN2: 211.2 ng
    MC2: 190.2 ng
    BN2:  184.4 ng
    BC2:  219.2 ng
  • Digestions were run on a 1% agarose gel at 125V for 30 minutes.

  1. Ladder
  2. MN2
  3. MC2
  4. BN2
  5. BC2
  6. Ladder


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