SBB10Ntbk-JennaKelleher

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3/3/2010

Gameplan

1. If ligations can sit over the weekend (ask GSI), start Ligation of EcoRI/BamHI digests for sbb28, sbb29 and then do Transformation by heat-shock. Otherwise, proceed to (2).
2. Perform Macherey-Nagel Miniprep for sbb42, either while waiting for ligations or on its own.
3. Ask about homework.

Lab Issues

sbb42 miniprep

centrifuged instead of vortexed, pipetted 42-3 up and down instead of vortexing (vortexed the rest after...)
42-4 DNA column seems sketchy... liquid making it to bottom of the tube before centrifuge
DNA sat in columns in between miniprep steps for a while... up to 5 minutes. Hopefully this isn't a problem.

sbb28 and sbb29 ligations

added LB (2YT) instead of KCM to cells before heat shocking - tossing samples and redoing ligation next week

What's Next

Monday, 3/8/2010

- Analytical Gel for sbb42: If it worked, the samples will be ready for sequencing
- Ligation and Transformation of sbb28 and sbb29

Wednesday, 3/11/2010

- Miniprep and Analytical Gel for sbb28 and sbb29, then they'll be ready for sequencing

3/1/2010

Performed EcoRI/BamHI transfer for sbb42, followed by ligation, transformation, and rescue.
- Incubation was started at 3:55pm - plating done by GSI's (thank you!)

Meanwhile, zymo'd sbb28 and sbb29 preperative gel products - ready for ligation/transformation next time.

2/24/2010

Labwork

Performed EcoRI/BamHI Digests on sbb28 and sbb29
After, ran 2 preparative gels

10 microliters digestion product
1 microliters undiluted 6x loading buffer

Gel Results

Success! Gel loaded and imaged by Andrew Saarni


Relevant Lanes:

1. Ladder
2. blank (ran off the gel...)
3. sbb28
4. sbb29

After, cut out bands and melted in 600 microliters of ADB buffer, stored for later use.

2/22/2010

Labwork

Last time: basic PCR for sbb28, sbb29
This time: ran 2 analytical gels

3 microliters PCR
7 microliters Loading Buffer

While waiting, ran zymo cleanup on the 2 samples, final volume of 30.6 microliters
Stored in 140L Box A

Next time: digestion

Gel Results

Analytical gel images shown below (ladder breakdown on left, gels on right), loaded and imaged by Dorothy Tulanont



Relevant Lanes:

1. ladder
2. sbb28 - looks to be about 1500 bp
3. sbb29 - looks to be about 1000 bp

Both of these values are close to desired lengths, so PCR worked!

2/17/2010

Labwork

Prepared sbb28 and sbb29 for PCR after creating a master mix with Jeni Lee
sbb28 in 2k55 and sbb29 in 55

Updated Constuction Files

Biobricking of 099 rep protein basic part sbb29

PCR repo99-F and JG002-R on pEC52 gene             (978 bp, EcoRI/BamHI)
Sub into pBca9523-Bca1144                           (EcoRI/BamHI, 910+2472, L)
Product is pBca9523-sbb29	{rbs.repO99!} 
----------------------------
repo99-F   Cloning of repo99   
ccaaaGAATTCatgAGATCTcctggaaggaatcgtaacac
JG002-R   Cloning of  O99 rep Gene   
CTGATGGATCCCTACTCTACAAGACCTCGTTTTttc

Biobricking of CA42 rep gene basic part sbb28

PCR repCA42-F and KRM002 on pEC22-CA42 gene      (1496 bp, EcoRI/BamHI)
Sub into pBca9523-Bca1144                           (EcoRI/BamHI, 910+2472, L)
Product is pBca9523-sbb28     {5'UTR.repCA42!} 
----------------------------
repCA42-F   Cloning of repCA42   
ccaaaGAATTCatgAGATCTcagctgaagtgaccggattag
KRM002   Reverse oligo for cloning of rbs.repCA42!    
ctgatGGATCCctattttccgcttttccag

Biobricking of pre-pro sequence basic part sbb42

Eco/Bam transfer pBjh1601AC-Bjh1723
Sub into pBjk2741-Bca1144            (EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-Bjh1723  	{rbs_pelB>}

2/10/2010

Project Information

29071-O99 rep Protein, CA42 rep Gene, Pre-Pro sequence

Construction Files

Jenna Kelleher 099 Rep Protein (sbb29)
Jenna Kelleher CA42 Rep Gene (sbb28)
Jenna Kelleher Pre-pro Sequence (sbb42)

Useful Protocols

For SBB28 and SBB29:
Cloning by PCR
Regular Zymo Cleanup

For SBB42:
Eco-Bam Transfer
Small-Frag Zymo Cleanup

For All 3:
EcoRI/BamHI Digest of PCR Products
Zymo Gel Purification
Ligation of EcoRI/BamHI digests
Transformation by heat-shock
Picking of colonies
Macherey-Nagel Miniprep
EcoRI/BamHI Digest of PCR Products
Analytical digests (Mapping)