Berk2010-Christoph

Christoph Neyer 21:26, 18 June 2010 (EDT)

Did Eco/Bam digest of good parts and here is the layout on the gel:

Lane #	Part
	A
1	\"7 with iGEM10_022
\"
3	\"9 with iGEM10_023
\"
5	\"9 with iGEM10_024
\"
7	\"9 with iGEM10_028
\"
9	4 with iGEM10_036
11	4 with iGEM10_037
13	\"4 with iGEM10_009
\"
15	\"9 with iGEM10_025
\"
	B
2	\"7 with iGEM10_022
\"
4	\"9 with iGEM10_023
\"
6	\"9 with iGEM10_024
\"
8	\"9 with iGEM10_028
\"
10	4 with iGEM10_036
12	4 with iGEM10_037
14	\"4 with iGEM10_009
\"
16	\"9 with iGEM10_025
\"
	A
17	\"9 with iGEM10_027
\"
19	\"8 with iGEM10_029
\"
21	\"9 with iGEM10_026
\"
23	\"9 with iGEM10_027
\"
	B
18	\"9 with iGEM10_027
\"
20	\"8 with iGEM10_029
\"
22	\"9 with iGEM10_026
\"
24	\"9 with iGEM10_027
\"

Christoph Neyer 15:51, 18 June 2010 (EDT)

Found out that we mixed up some things while creating the plate layouts for stage1 and we ended up plating on the wrong antibiotics.

• We need to spot check and colony PCR the catchups we started yesterday. (9 total):

Name	Strain	Lefty Input	Righty Input
5 with iGEM10_026	L	9 with 8	7 with 23
5 with iGEM10_027	L	9 with 20	7 with 2
5 with iGEM10_032	L	9 with 23	7 with 13
5 with iGEM10_034	L	9 with 20	7 with 5
5 with iGEM10_008	L	9 with 20	7 with 19
8 with iGEM10_035	L	6 with 13	4 with 9
5 with iGEM10_034	R	9 with 20	7 with 5
5 with iGEM10_008	R	9 with 20	7 with 19
8 with iGEM10_035	R	6 with 13	4 with 9

• Assembled these parts that we missed/plated on the wrong antibiotics manually:
  

Name	Strain	Lefty Input	Righty Input
5 with iGEM10_035	L	9 with 13	7 with 9
6 with iGEM10_033	L	8 with 4	9 with 3
9 with iGEM10_034	L	5 with 20	6 with 5
9 with iGEM10_008	L	5 with 20	6 with 19
5 with iGEM10_035	L	9 with 13	7 with 9
5 with iGEM10_038	R	9 with 11	7 with 24
7 with iGEM10_040	R	4 with Bjh1882	5 with 15
6 with iGEM10_033	R	8 with 4	9 with 3
9 with iGEM10_034	R	5 with 20	6 with 5
9 with iGEM10_008	R	5 with 20	6 with 19
5 with iGEM10_035	R	9 with 13	7 with 9

Christoph Neyer 13:30, 18 June 2010 (EDT)

We spot checked our colony PCR colonies on ACK to check for co-transformation and this is what our plates look like:
Plate A

and Plate B

Christoph Neyer 21:22, 17 June 2010 (EDT)

Colony PCR results for Stage1 parts:
Here are columns 1-6 in two parts:
Part 1 (Parts 1 and 2 are picutres of the same gels)

and Part 2

Here are columns 7-10:

Christoph Neyer 18:24, 17 June 2010 (EDT)

Most of the colonies turned out good for Stage 1 of the robot assembly. We picked colonies for colony PCR.

Manually made these parts for Stage 1 using the robot protocol. We missed them some how:

Name	Strain	Lefty Input	Righty Input
5 with iGEM10_026	L	9 with 8	7 with 23
5 with iGEM10_027	L	9 with 20	7 with 2
5 with iGEM10_032	L	9 with 23	7 with 13
5 with iGEM10_034	L	9 with 20	7 with 5
5 with iGEM10_008	L	9 with 20	7 with 19
8 with iGEM10_035	L	6 with 13	4 with 9
5 with iGEM10_034	R	9 with 20	7 with 5
5 with iGEM10_008	R	9 with 20	7 with 19
8 with iGEM10_035	R	6 with 13	4 with 9

Christoph Neyer 21:15, 16 June 2010 (EDT)

While plating Stage1 2ab assembly set A we may have reversed the order of Col 1 B (30 ul).
The cells from plate A3-D3 may be in wells B4-B1 instead of B1-B4 on the 2x12 plated for Col 1A from the reaction plate.
Also note rows A and B were switched on the plate for Col 5A as noted on the plate. A is 30ul and B is 60ul instead of the other way around.

Christoph Neyer 18:10, 16 June 2010 (EDT)

Redoing Stage1B and doing Stage1A 2ab assembly today.
Check autoclave at 4:30.
Decided to redo all of B along with A. We can do them together on the robot by doubling everything but changing the source plate Made dilution plate A, and added 60uL of dilution to dilution plate B. 7 with 11 still looked different Distributed digestion cocktail to both dilution plates Note: Robot begins pipetting air if it uses the same tip for two long. We stopped the program halfway through to change its tip Distributed Righty and Lefty parts to reaction plates. Made sure there were duplicate columns for parts being transformed twice Checked that at this point all wells had the same volume Put plates in thermocycler to incubate and heat kill Distributed antibiotics to 10 plates. All of LB agar was contaminated so will pour that during the rescue step added ligation mixture to each well and let incubate at room temperature.

Christoph Neyer 18:11, 16 June 2010 (EDT)

Robot 2AB assembly Stage 1

Yesterday we did stage 1 of our 2AB assembly only on plate B. Today we will repeat the process on plate A without making all of the mistakes that we did yesterday.

June 15:
Autoclaved our strips for plating. Learned how to work the autoclave machine

Made a new plate layout for the reaction plate. Wells are organized by what strain the part needs to be transformed into to
MISTAKE: Did not listen to Tim and organized wells by column instead of rows. Rows are better because the plating strips have 2 rows and 12 columns
MISTAKE: Did not read procedure all the way through in detail and assumed that we could use a well for two transformations. Our dilution and reaction plate needs to have duplicates of the parts that go into both Righty and Lefty strains. We have to add the duplicates in either the dilution or reaction plate
Made the dilution plate B (30ul of DNA into each well + 30ul of NEB2 stock diluted by a factor of 5). Dilution plate had the same layout as methylated stock plate so we did it by hand. Next time, we should consider adding duplicates of the parts being transformed twice.
NOTE: 7 with 11 was frozen in our methylated stocks plate. This is the part that was mini-prepped separately the day before. All other wells weren't frozen.

Had the robot make reaction plate (distribute digestion mastermix to all wells, distribute all Lefty parts, mix, distribute all righty parts, mix)
While the reaction plate incubated in the black thermocycler (which also did heat kill of the digestion enzymes) we distributed the antibiotics to the 24-well strips. We made antibiotics (dilute stock by 10, add 1ul of dye). This part went fine except we should have labeled the plates before we put them on the robot. Added LB agar to the plates and let them dry next to the flame.
MISTAKE: forgot to make second plate for parts being transformed twice. Column 5 in reaction plate needed to go to two different plates. We made the extra plate by hand

Had robot add the ligation cocktail to the wells and let it incubate at room temperature. We began thawing our bacteria.
Did transformation of bacteria. We had enough volume to seed at least 60uL of bacteria per column.
MISTAKE: Did not realize that the protocol was for seeding 10uL of bacteria. We wanted to seed about 150uL. We should not have done the centrifugation and aspiration of the supernatant step.
MISTAKE: ejecting tips from multi-channel before releasing cells on plate. We lost 60uL of our cells from column 7.
MISTAKE: We split the volume in column 5 into column 9 (because column 5 had to be transformed into both righty and lefty strains). Thus we also did not have enough volume for those parts.
MISTAKE: not taking a short break after lunch to re-energize and prevent making silly mistakes

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) Check
• Plate (1hr)Check

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:

Column 5 is Lefty and was transformed with only half the ligation product.
Column 9 is righty and was also transformed with half the ligation product.

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.