IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/08/06: Difference between revisions

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'''amiRNA'''
'''amiRNA'''
* amiRNA-transformed turbo cells didn't grow in YEB medium. There should be more LB+Amp available by the end of the day. In the meantime, we're digesting some more backbone (8x20ul X+P+B21)
* amiRNA-transformed turbo cells didn't grow in YEB medium. There should be more LB+Amp available by the end of the day. In the meantime, we're digesting some more backbone (8x20ul X+P+B21)
* ligations of Bet/LTP into V0120


'''ihpRNA'''
'''ihpRNA'''
*no colonies on plates from transformations done yesterday after 16 hours (we let these plates grow for longer)
*no colonies on plates from transformations done yesterday after 16 hours (we let these plates grow for longer)
*redoing ligations of ihpRNA inserts in V9/V10
*redoing ligations of ihpRNA inserts in V9/V10
**Redigested and gel purified some backbone
**Redigested and gel purified some backbone
[[Image: |240px|V9 and V10 backbone]]
 
''Concentrations:'' V9 (5 ng/uL); V10 (6 ng/uL)
 
**12 ligations each for V9 and V10


==Team Flavor==
==Team Flavor==
Line 21: Line 27:
''Note: both gels images are the same, but with different contrasts/resolutions''
''Note: both gels images are the same, but with different contrasts/resolutions''


# Ladder
# 1 kb Ladder (Invitrogen)
# 004-1
# 004-1
# 004-2
# 004-2
Line 31: Line 37:
====Analysis====
====Analysis====
Both the backbone and the insert in lane 1 look like the correct length (3.2 and 1.1 kb respectively).  Lane two consists of a backbone of the correct length, but the insert appears to be ~1.5 kb, too long for the J45004 BioBrick part.  Lane 3 appears undigested; we are uncertain as to why.
Both the backbone and the insert in lane 1 look like the correct length (3.2 and 1.1 kb respectively).  Lane two consists of a backbone of the correct length, but the insert appears to be ~1.5 kb, too long for the J45004 BioBrick part.  Lane 3 appears undigested; we are uncertain as to why.
===Transformation of Mira/Brazz StrepII constucts into E45===
*100-200 ng of construct DNA was transformed with 15μL of E45 expression ''E. Coli''; transformation procedure was followed; transformed cells were plated on LB+AMP and left to grow O/N
Transformations:
Mira N1
Mira N2
Mira C1
Mira C2
Brazz N1
Brazz N2
Brazz C1
Brazz C2
==Team Fence==
===Digestions===
pACT2
*1μL Bam
*1μL Xho
*6μL pACT2 (tube with a green top)
*1μL FD Green buffer
*1μL DH<sub>2</sub>O
RXRLc
*1μL Bam
*1μL Xho
*9μL RXRLc4
*2μL FD Green buffer
*7μL DH<sub>2</sub>O
Barstar
*1μL Eco
*1μL Pst
*2μL Barstar 3
*1μL FD Green buffer
*5μL DH<sub>2</sub>O
Barnase
*1μL Eco
*1μL Pst
*1.5μL Barnase 2
*1μL FD Green buffer
*5.5μL DH<sub>2</sub>O
5xGalUAS
*1μL Spe
*1μL Pst
*2μL 5xGalUAS (from Karmella)
*1μL FD Green buffer
*5μL DH<sub>2</sub>O
pENT Cup
*1μL Spe
*1μL Pst
*1μL pENT Cup
*1μL FD Green buffer
*6μL DH<sub>2</sub>O
EcR-Gal
*1μL Xba
*1μL Pst
*2μL EcR-Gal 7
*1μL FD Green buffer
*5μL DH<sub>2</sub>O
VP16
*1μL Spe
*1μL Pst
*5μL VP16(1)
*1μL FD Green buffer
*2μL DH<sub>2</sub>O
RXRLc
*1μL Xba
*1μL Pst
*9μL RXRLc4
*2μL FD Green buffer
*7μL DH<sub>2</sub>O
V24
*1μL Eco
*1μL Pst
*3μL V24
*1μL FD Green buffer
*4μL DH<sub>2</sub>O
V26
*1μL Eco
*1μL Pst
*9μL V26
*12μL FD Green buffer
*7μL DH<sub>2</sub>O
PhyB
*1μL Bam
*1μL Nco
*16μL PhyB
*2μL FD Green buffer




Revision as of 15:02, 7 September 2010

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Team Allergy

amiRNA

  • amiRNA-transformed turbo cells didn't grow in YEB medium. There should be more LB+Amp available by the end of the day. In the meantime, we're digesting some more backbone (8x20ul X+P+B21)
  • ligations of Bet/LTP into V0120

ihpRNA

  • no colonies on plates from transformations done yesterday after 16 hours (we let these plates grow for longer)
  • redoing ligations of ihpRNA inserts in V9/V10
    • Redigested and gel purified some backbone

[[Image: |240px|V9 and V10 backbone]]

Concentrations: V9 (5 ng/uL); V10 (6 ng/uL)

    • 12 ligations each for V9 and V10

Team Flavor

Confirmation Digest of J45004

  • J45004 in V0120 backbone; digested ~500ng DNA with Xba1/Pst1


Note: both gels images are the same, but with different contrasts/resolutions

  1. 1 kb Ladder (Invitrogen)
  2. 004-1
  3. 004-2
  4. 004-3
  5. Ladder

Backbone: 3.2 kb; Insert: 1.1 kb

Analysis

Both the backbone and the insert in lane 1 look like the correct length (3.2 and 1.1 kb respectively). Lane two consists of a backbone of the correct length, but the insert appears to be ~1.5 kb, too long for the J45004 BioBrick part. Lane 3 appears undigested; we are uncertain as to why.

Transformation of Mira/Brazz StrepII constucts into E45

  • 100-200 ng of construct DNA was transformed with 15μL of E45 expression E. Coli; transformation procedure was followed; transformed cells were plated on LB+AMP and left to grow O/N

Transformations: Mira N1 Mira N2 Mira C1 Mira C2 Brazz N1 Brazz N2 Brazz C1 Brazz C2


Team Fence

Digestions

pACT2

  • 1μL Bam
  • 1μL Xho
  • 6μL pACT2 (tube with a green top)
  • 1μL FD Green buffer
  • 1μL DH2O

RXRLc

  • 1μL Bam
  • 1μL Xho
  • 9μL RXRLc4
  • 2μL FD Green buffer
  • 7μL DH2O

Barstar

  • 1μL Eco
  • 1μL Pst
  • 2μL Barstar 3
  • 1μL FD Green buffer
  • 5μL DH2O

Barnase

  • 1μL Eco
  • 1μL Pst
  • 1.5μL Barnase 2
  • 1μL FD Green buffer
  • 5.5μL DH2O

5xGalUAS

  • 1μL Spe
  • 1μL Pst
  • 2μL 5xGalUAS (from Karmella)
  • 1μL FD Green buffer
  • 5μL DH2O

pENT Cup

  • 1μL Spe
  • 1μL Pst
  • 1μL pENT Cup
  • 1μL FD Green buffer
  • 6μL DH2O

EcR-Gal

  • 1μL Xba
  • 1μL Pst
  • 2μL EcR-Gal 7
  • 1μL FD Green buffer
  • 5μL DH2O

VP16

  • 1μL Spe
  • 1μL Pst
  • 5μL VP16(1)
  • 1μL FD Green buffer
  • 2μL DH2O

RXRLc

  • 1μL Xba
  • 1μL Pst
  • 9μL RXRLc4
  • 2μL FD Green buffer
  • 7μL DH2O

V24

  • 1μL Eco
  • 1μL Pst
  • 3μL V24
  • 1μL FD Green buffer
  • 4μL DH2O

V26

  • 1μL Eco
  • 1μL Pst
  • 9μL V26
  • 12μL FD Green buffer
  • 7μL DH2O

PhyB

  • 1μL Bam
  • 1μL Nco
  • 16μL PhyB
  • 2μL FD Green buffer



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