IGEM:UC Berkeley/2006/bryans notebook

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08/10/06

• miniprepped pJ23006-J23058-1, pJ23006-J23022-1, pJ23006-J23033-D, pJ23006-J23033-E, pJ23006-J23033-E, pJ23006-J23033-F, pJ23006-J23034-D, pJ23006-J23034-E, pJ23006-J23034-F.
• transformed pJ23006-J23022-1 into 1122s.
• digested pJ2306-J23058-1 with BamHI/HindIII

--Bryanh 03:54, 9 August 2006 (EDT)

1.pJ23006-J23009-1 assay
2.Lock3 Variants
3.pK2, pK3--1129 assays
4.QPCR Experiment
5.key3c-key3d juxtaposation
6.Key3d loop adjustments
7.key3d-TT construct

1.

• discuss sequence results with chris

2.

• update map for pSB3C6-J23018 according to new sequence data.
• miniprepped pSB3C6-J23031-1, pSB3C6-J23032-1, pSB3C6-J23036-1
• picked colonies for pSB3C6-J23031-1, pSB3C6-J23032-1, pSB3C6-J23036-1 along with J01122 to compare.
• investigate the locks that didnt clone correctly.

3.

• colony PCR off cultures.

For pSB1A2-J23041 and pSB1A2-J23042: ca998/G00101

clone: 343bp
parent(pSB1A2-J01007):238bp
parent(pJ23006-J23008):474bp

all cultures were perfect. miniprep pSB1A2-J23041-1 and pSB1A2-J23042-1

• miniprep the good ones.
• transform into 1122

4.

• fix KB oligo/QPCR oligo naming overlap.

5.

• resequenced pJ23006-J23044-1 using this time G00101
• picked a colony to grow and tecan

6.

• grew up one green colony.

7.

• colony PCR a few colonies of this using ca998/G00101 as the primers and pJ23006-J23009 as the parent control.

clone: 692bp
parent(pJ23006-J23009): 345bp
parent(pSB1AK3-B0015): 388bp

all colonies were perfect.

• grew up colony of pJ23006-J23022-1.

Bryanh: 08/08/06

1. pJ23006-J23009-1 assay
2. Lock3 Variants
3. pK2, pK3--1129 assays
4. QPCR Experiment
5. key3c-key3d juxtaposation
6. Key3d loop adjustments
7. key3d-TT construct

• jen sequenced pSB3C6-bca1025 with ca1001 and filled out the sequence log as "kad"

1. enter sequencing results.
2.

• miniprep pSB3C6-J23031-1, pSB3C6-J23032-1, pSB3C6-J23036-1;
• miniprepped pJ23006-J23030-B,C, pJ23006-J23033-A,B,C, pJ23006-J23034-A,B,C, pJ23006-J23035-A,B,C. test digested with XbaI/AlwNI. for a successful sublcone i expect to see bands of ~600, ~1700bp. a singley cut plasmid results in a band of ~2300bp. one band at about 1150bp is doubley cut where both bands are on top of each other.
  
• transformed pSB3C6-J23031-1, pSB3C6-J23032-1, pSB3C6-J23036-1

3. picked colonies of pSB1A2-J23041 and pSB1A2-J23042
4. hold off
5.

• miniprepped and sequenced pJ23006-J23043.1 and pJ23006-J23044.1
• transformed into 1122

6.

• miniprepped pJ23006-J23009-1. Digested pG80-GFPmut3 (BamHI/HindIII, Neb2, small-887bp),

Pasted into pJ23006-J23009-1 (BamHI/HindIII, Neb2, large-2323bp).

• transformed

7. digested, ligated, and transformed key3d with b0015 according to the construct file pJ23006-J23022.

--Bryanh 18:46, 7 August 2006 (EDT)

1. pJ23006-J23009-1 assay
2. Lock3 Variants
3. pK2, pK3--1129 assays
4. QPCR Experiment
5. key3c-key3d juxtaposation

1. analyze sequence data

2.

• grew up clones that were correct according to the sequencing. pJ23006-J23031-1, pJ23006-J23032-1, pJ23006-J23036-1 were correct.
• picked 3 new colonies for pJ23006-J23030-1, pJ23006-J23033-1, pJ23006-J23034-3, pJ23006-J23035-1. these will be miniprepped and then mapped with XbaI/AlwNI. if bad then we expect to only see one fragment because XbaI is deleted, among other things.

3. miniprepped and digested pJ23006-J23008 and then ligated with the J01007/J01006 digests. transformed.

4. hold off.
5. Colony PCR 23043.1-43.4 and 23044.1-44.4 is ca998 and g00101. one control using same oligos but 23006-23008 as a template. all of these came out looking the right size.

• pick a colony from the first of each colony.

8-4-06
hey brian.
we ligated and transformed J23043 and J23044 for you today. we couldn't make J23021 and J23042 though with the J01006 and J01007 digests because there wasn't a J23008 Alwn/Xbal digest, only a J23008 Alwn/Spe digest and there was not enough of the J23008 miniprep to make a new digest, so we grew up J23008 from the -80 instead. Also, you guys are running low on J23009 but didn't have a minus 80 stock of it, so me and matt transformed the miniprep that was sequenced in TGIs.
-Samantha

Hey Brian. This is what I did for you today:

• religated key3 variants with pSB3C6-J23018 (it wouldnt have worked to plate your ligation/transformations becuase they sat in the shaker over night)
• transformed and plated on CAM plates
• made names for all 7 of these constructs and temporary construction files (need to be formatted correctly. i just put into in there so we know what they are)
• checked sequencing results, threw out transformations i did earlier of pJ23006-J23035-1, pJ23006-J23034-3, pJ23006-J23033-1, and pJ23006-J23030-1. picked 3 new colonies of each of those.

--Bryanh 20:54, 3 August 2006 (EDT)

1. pJ23006-J23009-1 assay
2. Lock3 Variants
3. pK2, pK3--1129 assays
4. QPCR Experiment
5. key3c-key3d juxtaposation

1.

• tecan assay:

	199  	287  	431  	22903  	1388  	1309  	867  	973  	1214

TGI, 1122, 1122x1129, 1022, pJ23006-J23009-1-E, pJ23006-J23009-1-F, pJ23006-J23009-1-G, pJ23006-J23009-1-H, pJ23006-J23009-1-I

• sent for sequencing again using the reverse sequencing oligo G00101.

2.

 Digested the lock3variant PCRs and ligated with the 23018 digest.

• transformed but not plated. the cells are in the rescue phase incubating in LB. these just need to be plated on chloramphenicol now.
• sent pSB3C6-J23018 for sequencing off the midiprep.

3.

• fixed construction error. experiment needs to be restarted.

4.

• hold off for now.

5.

• digested pJ23006-J23008 and pJ23006-J23009 according to the construct files for pJ23006-J23043 and pJ23006-J23044.

i gel purified these digests so that they are ready to be ligated as is. this means that the Digest pJ23006-J23008 (AlwNI/SpeI, Neb2, large-1826bp) and Digest pJ23006-J23009 (AlwNI/XbaI, Neb2, small-652bp are cleaned up and mixed together and ready to be ligated to make pJ23006-J23043. likewise the pJ23006-J23044 construct is in the same form. these ependorfs are labeled as "pJ23006-J23043 dig. C" and "pJ23006-J23044 dig. C" and are sitting in the riboregulator's oligonucleotide box (since this is the only one with room.)

--Bryanh 23:54, 1 August 2006 (EDT) experiments

1. pJ23006-J23009-1 assay
2. Lock3 Variants
3. pK2, pK3--1129 assays
4. QPCR Experiment

1.assayed: <> 6 7 8 9 10
A 257 444 729 2287 56416

TGI, 1122, 1122x1129, pJ23006-J23009-1x1122, 1022
These results are somewhat unexpected compared to yesterday's assay which was pretty much the same thing except we have 1022 data on this one.

• due to the variation in flouresence the experiment will be repeated using several different colonies in an effort to establish relatively presice numbers.

2. minipreped pSB3C6-J230181,2. digested EcoRI/XbaI. the gel came out really bad due to the low copy number of the p15a backbone and the digest of the 23006-lock3variants also came out really bad because the fragment was so small it wasnt visible on the gel (~260). as such, a pcr strategy will be taken to amplify the small fragment using the pSB1A2 sequencing oligos (ca998 and G00101) and then digest from there. the pcr has been done. in order to get the pSB3C6-J23019-1 backbone digested we will need to midiprep it to obtain a high concentration of the low copy plasmid and then the digest should come out better.

• a 50ml solution of pSB3C6-J23018 has been grown up for the midi.
• sent one of each 23006-lock3variant for sequenceing.

3. grew up two colonies of each pSB1A2-J230024,-J23025

--Bryanh 31 July 2006 (EDT)
experiments

1. pJ23006-J23009-1 assay
2. Lock3 Variants
3. pK2, pK3--1129 assays
4. QPCR Experiment

1. assay: 210, 449, 248, 312 (TGI, 1122, 1122x1129, pJ23006-J23009-1)
2. grow up pSB3C6-J23018. screen pJ23006-Lock3Variants. miniprep them.
PCR Screen:

 pJ23006-J23030: KB014/Bba_G0010 expected band size: ~140
 pJ23006-J23031: KB014/Bba_G0010 expected band size: ~140
 pJ23006-J23032: KB014/Bba_G0010 expected band size: ~140
 pJ23006-J23033: KB018/Bba_G0010 expected band size: ~140
 pJ23006-J23034: KB020/Bba_G0010 expected band size: ~140
 pJ23006-J23035: KB021/Bba_G0010 expected band size: ~140
 pJ23006-J23036: KB022/Bba_G0010 expected band size: ~140

3. digested J01007, J01006 with AlwnI/SpeI and ligated with pSB1A2-J01129.transformed.
4. oligos arrived

for monday:

1.grow up colonies.
2. colony PCR pJ23006-lock3Variants and then miniprep the good ones. digest EcoRI/SpeI (this doesnt have to be done on this day.)
3. miniprep J01007, J01006. digest and ligate into pSB1A2-J01129. tranform.

--Bryanh 15:22, 27 July 2006 (EDT)
experiments

1. pJ23006-J23009-1 assay
2. Lock3 Variants
3. pK2, pK3--1129 assays
4. QPCR Experiment

1. TGI, 1122, 1122x1129, pJ23006-J23009-1 plates are sitting in the fridge.
2. grew up four colonies of each pJ23006-Lock3Variant to colony PCR (or miniprep PCR) them on monday.
3. minipreped J01007, J01006. Digestion coctail is ready to go and is sitting in the fridge, just add enzymes (AlwnI and SpeI when its here.)
4. waiting on oligos

--Bryanh 15:24, 26 July 2006 (EDT)
experiments

1. pJ23006-J23009-1 assay
2. Lock3 Variants
3. pK2, pK3--1129 assays
4. QPCR Experiment

1.

• pJ23006-J23009-1 x J01122 is sitting on a plate.
• transformed 1122x1129 and plated TGI and pSB3C6-J01122.

2.

• grew two colonies of pSB1A2-J23018
• Klenow extension of lock3 variants:

1. J23030: KB014/KB015
2. J23031: KB014/KB016
3. J23032: KB014/KB017
4. J23033: KB018/KB019
5. J23034: KB020/KB019
6. J23035: KB021/KB019
7. J23036: KB022/KB019

3. J01007, J01006 cultures are growing up.

4. ordered oligos for QPCR --Bryanh 15:22, 27 July 2006 (EDT)