Berk2010-Christoph: Difference between revisions

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*Reorganize destination plate based on L and R not on antibiotics. CHECK
*Reorganize destination plate based on L and R not on antibiotics. CHECK
*AUTOCLAVE 24 well Strips! CHECK
*AUTOCLAVE 24 well Strips! CHECK
*Digesting (1hr)
*Digesting (1hr) Check
**Dilute NEB2 and DNA.  
**Dilute NEB2 and DNA.  
*Make plates (2hr)
*Make plates (2hr) Check
**Cherry pick antibiotics
**Cherry pick antibiotics Check
*Heat kill (20 mins)
*Heat kill (20 mins)Check
*Ligation (30 mins)
*Ligation (30 mins)Check
*Transform + Rescuing (1hr)
*Transform + Rescuing (1hr) Doing
*Plate (1hr)
*Plate (1hr)
Forgot to make two copies of the parts that are both lefty and righty. So, we just used half of ligation product of each. <br>
Here is the layout of the transformation plate:
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Stage1 Reaction Plate'''
| align="center" style="background:#f0f0f0;"|'''For plate B only!'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
|-
| A||rbs_phiC31>} = CA||||Promoter_rbs_GenRTn5 3\'TR = CA||||b1006Tn5 5\'TR = AC||||Promoter_rbs_GenRTn5 3\'TR = CA
|-
| B||{rbs_pelB>}} = CA||||Pbad{rbs_pelB>} = CA||||Promoter_rbs_GenRsleeping_beauty_3\'TR = CA||||b1006sleeping_beauty_5\'TR = AC||||Promoter_rbs_GenRsleeping_beauty_3\'TR = CA
|-
| D||{rbs_pelB>}} = CA||||Promoter_rbs_GenRpiggieBac3\'TR = CA||||{} = CK||||Promoter_rbs_GenRsleeping_beauty_3\'TR = CA||||} = CK||||} = KC||||} = KC||||{{


==[[User:Christoph Neyer|Christoph Neyer]] 21:29, 14 June 2010 (EDT)==
==[[User:Christoph Neyer|Christoph Neyer]] 21:29, 14 June 2010 (EDT)==

Revision as of 17:13, 15 June 2010

Christoph Neyer 20:10, 15 June 2010 (EDT)

  • Reorganize destination plate based on L and R not on antibiotics. CHECK
  • AUTOCLAVE 24 well Strips! CHECK
  • Digesting (1hr) Check
    • Dilute NEB2 and DNA.
  • Make plates (2hr) Check
    • Cherry pick antibiotics Check
  • Heat kill (20 mins)Check
  • Ligation (30 mins)Check
  • Transform + Rescuing (1hr) Doing
  • Plate (1hr)

Forgot to make two copies of the parts that are both lefty and righty. So, we just used half of ligation product of each.
Here is the layout of the transformation plate:

' Stage1 Reaction Plate For plate B only! ' ' ' ' ' ' '
A rbs_phiC31>} = CA Promoter_rbs_GenRTn5 3\'TR = CA b1006Tn5 5\'TR = AC Promoter_rbs_GenRTn5 3\'TR = CA
B {rbs_pelB>}} = CA Pbad{rbs_pelB>} = CA Promoter_rbs_GenRsleeping_beauty_3\'TR = CA b1006sleeping_beauty_5\'TR = AC Promoter_rbs_GenRsleeping_beauty_3\'TR = CA
D {rbs_pelB>}} = CA Promoter_rbs_GenRpiggieBac3\'TR = CA {} = CK Promoter_rbs_GenRsleeping_beauty_3\'TR = CA } = CK } = KC } = KC {{

Christoph Neyer 21:29, 14 June 2010 (EDT)

Goal for tomorrow is to transform and plate stage 1A and 1B. That involves:

  • Reorganize destination plate based on L and R not on antibiotics.
  • AUTOCLAVE 24 well Strips!
  • Digesting (1hr)
    • Dilute NEB2 and DNA.
  • Make plates (2hr)
    • Cherry pick antibiotics
  • Heat kill (20 mins)
  • Ligation (30 mins)
  • Transform + Rescuing (1hr)
  • Plate (1hr)

Christoph Neyer 14:18, 14 June 2010 (EDT)

Thanks to Tim and Shelly for picking colonies from methylation transformation

  • May have mislabeled these tubes, so we are running a BglII/Xho map:

Put in digest at 11am. Run gel at 11:30.

Looks ok. Added these parts to methylated parts plates

Lane # ID Name Expected fragments '
1 4 with 10 #1 pMLL4-AC+B10sbb43 850 1990
2 4 with 10 #2 pMLL4-AC+B10sbb43 850 1990
3 4 with 18 #1 pMLL4-AC+B10sbb13 850 2160
4 4 with 18 #2 pMLL4-AC+B10sbb13 850 2160
5 4 with 9 #1 pMLL4-AC+B10Sbb20 850 2450
6 4 with 9 #2 pMLL4-AC+B10Sbb20 850 2450
7 9 with 3 #1 pMLL9-CA+Bjh1853 300 1500
8 9 with 3 #2 pMLL9-CA+Bjh1853 300 1500

Christoph Neyer 21:36, 11 June 2010 (EDT)

Plated Transformations from robot dilution.

Christoph Neyer 19:04, 11 June 2010 (EDT)

Next step is to transform each part into a Righty or Lefty strain.
Manually transformed parts from dilution plate into Righty and Lefty strains. Incubated at 5:10. Take out at 5:50.
Transformation plate has same layout as dilution plate below. Here is layout again:

Transformation plate layout

Columns 1-6 are in Righty strains. Columns 7-12 are in Lefty strains.

' 1 2 3 4 5 6 7 8 9 10 11 12
A 4 with 16 5 with 20 6 with 12 7 with 10 8 with 17 9 with 11 4 with 10 6 with 11 6 with 5 7 with 13 8 with 1 9 with 3
B 4 with 17 5 with 23 6 with 13 7 with 11 8 with 18 9 with 13 4 with 18 6 with 12 6 with 7 7 with 14 8 with 21
C 4 with Bjh1882 5 with 25 6 with 22 8 with 4 9 with 20 4 with 9 6 with 19 6 with Bjh2245 7 with 19 8 with 6
D 5 with 3 6 with 23 9 with 23 6 with 2 7 with 2
E 5 with 8 6 with 7 9 with 8 6 with 22 7 with 23
F 6 with Bca1091 6 with 23 7 with 24
G 5 with 15 6 with 24 7 with 5
H 5 with 3 6 with 4 7 with 9

Christoph Neyer 16:31, 11 June 2010 (EDT)

Did a test run with the robot using our dilution csv file. Checked cloumn 4 it worked well.
Miniprepped pMLL5+Bca1152 and sent in for sequencing as iGem021.
Using robot to dilute 4ul of each vector+part stage 0 assembly into 76ul of water. See this csv file: Media:Stage0PartsDilution.csv

Source plate layout:

' 1 2 3 4 5 6 7 8 9 10 11 12
A 6+2 6+11 6+22 7+9 7+24 5+23 8+17 4+10 9+3 9+20 7+24
B 6+2 6+11 6+22 7+10 5+3 5+23 8+17 4+10 9+3 9+20 9+11
C 6+4 6+12 6+23 7+11 5+3 8+1 8+18 4+16 9+8 9+23 9+23
D 6+4 6+12 6+23 7+13 5+8 8+1 8+18 4+16 9+8 9+23 pMLL5+Bca1091 (L)
E 6+5 6+13 6+24 7+14 5+8 8+4 8+21 4+17 9+11 pMLL4+Bjh1882 (L)
F 6+5 6+13 6+24 7+19 5+15 8+4 8+21 4+17 9+11 pMLL6+Bjh2245
G 6+7 6+19 7+2 7+23 5+20 8+6 4+9 4+18 9+13 6 +24 6+12
H 6+7 6+19 7+5 7+24 5+20 8+6 4+9 4+18 9+13 7 + 23 5+25 (D)

Destination plate layout:

Columns 1-6 need to be transformed into Righty strains. Columns 7-12 need to be transformed into Lefty strains.

' 1 2 3 4 5 6 7 8 9 10 11 12
A 4 with 16 5 with 20 6 with 12 7 with 10 8 with 17 9 with 11 4 with 10 6 with 11 6 with 5 7 with 13 8 with 1 9 with 3
B 4 with 17 5 with 23 6 with 13 7 with 11 8 with 18 9 with 13 4 with 18 6 with 12 6 with 7 7 with 14 8 with 21
C 4 with Bjh1882 5 with 25 6 with 22 8 with 4 9 with 20 4 with 9 6 with 19 6 with Bjh2245 7 with 19 8 with 6
D 5 with 3 6 with 23 9 with 23 6 with 2 7 with 2
E 5 with 8 6 with 7 9 with 8 6 with 22 7 with 23
F 6 with Bca1091 6 with 23 7 with 24
G 5 with 15 6 with 24 7 with 5
H 5 with 3 6 with 4 7 with 9

Christoph Neyer 13:57, 11 June 2010 (EDT)

  • Added grown up Lefty and Righty strains to 500ml each of LB. Growing up for 2-3hrs. Check at 1pm.
  • Running colony PCR gel for Bca1152+pMLL5. Expected band at ~260.


Colonies A,C,D look good. Chose C and D to miniprep. Cells were put in warm room at 9am.
Grab Bca1152 colony PCR colonies at 1 or 2pm.

  • Added missing parts to iGEM10 plate1 minis:
' 1 2 3 4 5 6 7 8 9 10 11 12
A 6+2 6+11 6+22 7+9 7+24 5+23 8+17 4+10 9+3 9+20 7+24 Lysis Device
B 6+2 6+11 6+22 7+10 5+3 5+23 8+17 4+10 9+3 9+20 9+11 5+25
C 6+4 6+12 6+23 7+11 5+3 8+1 8+18 4+16 9+8 9+23 9+23
D 6+4 6+12 6+23 7+13 5+8 8+1 8+18 4+16 9+8 9+23 pMLL5+Bca1091 (L)
E 6+5 6+13 6+24 7+14 5+8 8+4 8+21 4+17 9+11 pMLL4+Bjh1882 (L)
F 6+5 6+13 6+24 7+19 5+15 8+4 8+21 4+17 9+11 pMLL6+Bjh2245
G 6+7 6+19 7+2 7+23 5+20 8+6 4+9 4+18 9+13 6 +24 6+12
H 6+7 6+19 7+5 7+24 5+20 8+6 4+9 4+18 9+13 7 + 23

Christoph Neyer 21:03, 10 June 2010 (EDT)

TO DO:

  • Pick colonies from Bca1152 and do colony PCR. Then miniprep if time.
  • Play with robot
  • Make competent cells.
  • Transfer box parts to dilutions plate.
  • When oligos arrive. Attempt biobrick on Bjh2342CA.

Christoph Neyer 13:38, 10 June 2010 (EDT)

  • Had to re-grow Lefty and Righty strains since no colonies grew.
  • Need to make part 5 with 25 (pMLL5-CK+B10sbb37) or (pMLL5-CK+Bca1152)

Currently in DH10B. Need to Eco/Bam transfer it into pMLL5-CK.
Put E/B digest of Bca1152 in DH10B into thermocycler at 12:10pm. Take out at 1:10pm and do small fragment zymo cleanup.
Set up ligation of Bca1152 and pMLL5 at 2:15. Transform at 2:45.
Heat-shock transformed Bca1152+pMLL5 into Jtk049. Plate at 4:15pm.
Plated Bca1152+pMLL5 into Jtk049. Growing up over night.
Sent in iGEM020 for sequencing(6 with 12C from working box to double check).

Christoph Neyer 20:31, 9 June 2010 (EDT)

TO DO List:

  • Finish generating competent cells for "Lefty" and "Righty" strains.
  • Analyze sequencing data for pMLL6+Bjh2245 B and C.
  • Transform pMLL6+Bjh2245 into "Righty" strain.
  • Transform other parts into the correct "Righty" or "Lefty" strains to prepare for robot 2ab assembly.

Christoph Neyer 15:16, 9 June 2010 (EDT)


Colony PCR of pMLL6+Bjh2245 should be at about 265bps. Looks good. Wait for colonies to grow and then miniprep.

  • Sent in miniprepped pMLL4-Bjh1882 (D) and pMLL5-Bca1091 (2A) for sequencing.
  • Started growing up Lefty and Righty strains. Picked two colonies of each lefty and righty and grew them up in 10ml LB.
  • Designed three potential oligos for biobricking part Bjh2341CA (igemTen011, igemTen012, and igemTen013)
  • Miniprepped pMLL6+Bjh2245. Need to transform into "Righty strain"
  • Sent in minprepped pMLL6+Bjh2245 to be sequenced.

Christoph Neyer 13:28, 9 June 2010 (EDT)

Bca1091 is 60bps.
Colony PCR band for parts:

  • pMLL4+bjh1882 = 849bps
  • pMLL6+bjh2245 = 265bps
  • pMLL5+bca1091 = 228bps
  • For First try Colony PCR of 1882 and 1091:
    • Bca1091 pick lanes 8 and 10 for miniprepping.(Block #1: Colonies from well 1D and 2A)


  • For Second try Colony PCR of 1882 and 1091
    • Bjh1882 pick lanes 1 and 4 for miniprepping. (Block #2: Colonies from well A and D)

Christoph Neyer 19:37, 8 June 2010 (EDT)

TO DO Tomorrow:

  • Miniprep colonies that we pick for pMLL4+Bjh1882 and pMLL5+Bca1091. These are methylated correctly for the robot.
  • Colony PCR pMLL6+Bjh2245 in Jtk049. These are not methylated correctly.

Christoph Neyer 19:51, 8 June 2010 (EDT)

  • Ran gel of colony PCR pMLL4-Bjh1882 and pMll5-Bca1091 AGAIN:

ladder 1882a b c d 1901a(1) b c d ladder 1901a(2) b c d

Christoph Neyer 16:59, 8 June 2010 (EDT)

  • Ligated pMLL6 Eco/Bam digest with part Bjh2245.
  • Ran gel of colony PCR pMLL4-Bjh1882 and pMll5-Bca1091:

ladder 1882a b c d 1901a(1) b c d ladder 1901a(2) b c d
  • Repicking colonies since Colony PCR appeared contaminated. Block has same layout, shown below:
PCR Block 1882 and 1091 #2 ' ' ' '
1882 1091 #1 1091 #2
A A A
B B B
C C C
D D D
  • Ran out of "Righty" cells. So we are transforming Jtk049 with our E/B ligation of pMLL6+Bjh2245.

Christoph Neyer 14:04, 8 June 2010 (EDT)

  • Digested pMLL6 w/ Eco/Bam and gel purified.
  • Colony PCR of transformed pMLL4-Bjh1882 and pMLL5-Bca1091:
PCR Block 1882 and 1091 #1 ' ' ' '
1882 1091 #1 1091 #2
A A A
B B B
C C C
D D D

Christoph Neyer 19:30, 7 June 2010 (EDT)

Bjh1882 Vectors: AC
Bjh2245 Vectors: KA
Bca1091 Vectors: CK
pMLL4-AC
pMLL5-CK
pMLL6-KA
pMLL7-AK
pMLL8-KC
pMLL9-CA
Bjh1882+pMLL4-AC = Lefty
Bjh2245+pMLL6-KA = Righty
Bca1091+pMLL5-CK = Lefty
Ligated isolated Bjh1882 and Bca1091 into above vectors. Transformed into Lefty strains.
Need to ligate Bjh2245 (Missing pMLL6, need to digest).
TO DO:

  • Digest pMLL6 with Eco/Bam and gel purify. Then ligate with Bjh2245.
  • Check transformed Bjh1882 and Bca1091.
  • Transform ligated Bjh2245+pMLL6 into Righty strain

Christoph Neyer 16:35, 7 June 2010 (EDT)

Couldn't see fragments. Turns out Bjh2245 is 97bp and Bjh1882 is ~680bps.
Re-cut Bjh2245 and Bjh1882.
Small fragment zymo cleanup of Bjh2245 digest.


Gel purified Bjh1882.

Christoph Neyer 15:54, 7 June 2010 (EDT)

Eco/Bam digest of parts: Bjh2245 (LifeACT), Bjh1882 (RFP), and BCA1091 (Ptet).
Gel purified Bjh1882 and Bjh2245.
Small fragment zymo cleanup of BCA1091.

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