SBB09Ntbk-Jennifer Brophy

Jennifer Brophy 00:03, 9 February 2009 (EST)

If possible, make two circularly permutated ompG proteins. One with the backbone opening in loop 2 (aa 59-60) and one in loop 6 (somewhere within aa 220-231) where the residues of the mature protein are not visible in electron density maps, presumably because they are disordered.

to do

Jennifer Brophy 11:59, 9 February 2009 (EST)

Created Construction files for CPG_L2 and CPG_L6. Entered oligos in oligo log

BLAST oligos, enter parts in parts log, look into Nterm and Cterm linker sequences.

Jennifer Brophy 13:19, 18 February 2009 (EST)

Reconstitute oligos. 100uM by adding 10X nmol.

PCR of M10006 (<N.CPG_L2!, regular part, 211bp), A-M10007 (371bp), B-M10007 (343bp), A-M10009 (548bp), B-M10009 (175bp), M10010 (<C.CPG_L6>, regular part, 202bp).

zymo clean-up, PCR A+B for M1007 and M1009.

Jennifer Brophy 21:02, 19 February 2009 (EST)

Did zymo clean up of PCR products (small fragment and regular clean up where appropriate). Diluted some clean ups in to much water so i either redid the PCR or added more template for the A+B reactions. Did PCR A+B of M10007 (694bp) and M10009 (703bp). BUT THESE DIDNT WORK BECAUSE I FORGOT TO DO GEL PURIFICATION TO GET RID OF TEMPLATE DNA.

Jennifer Brophy 18:57, 20 February 2009 (EST)

Did gel purification of M10006,M10007A, M10007B, M10009A (no 9B because i had messed up the zymo clean up yesterday), and M100010. Failure to remove the genomic DNA via gel purification yesterday rendered the PCR of M10007 and M10009 useless.

Gel=ladder, M10006 (211bp), M10007A (371bp), M10007B (341bp), M10009A (548bp), M10010 (202bp).
Re-did PCR of M10009B.

gel purify M10009B. do next round (A+B) PCR. gel purify? start SOEing

Jennifer Brophy 13:07, 23 February 2009 (EST)

Zymo small fragment clean up of M10009B. Gel purify M10009B.

Gel= ladder, M10009B (175bp).
Do A+B PCR to get M10007 (694bp) and M10009 (703bp).

gel= ladder, M10007, M10009. Careful gel purification of M10007, less of a product, mixture of oligos?
Set up PCR of M10008A/B and M10011A/B.

Jennifer Brophy 12:23, 24 February 2009 (EST)

Gel purify M10008A (209bp), M10008B (701bp), M10011A (701bp), M10011B (209bp).

Gel= ladder, M10008A, M10008B, M10011A, M10011B.
Set up PCR for M10008 and M10011.

to do

Jennifer Brophy 13:27, 25 February 2009 (EST)

Gel purify M10008 (892 bp) and M10011 (892bp).

Gel= ladder, M10008, M10011.
Did Eco/Bam digest and ligation into pBca9495KC. Plated on KC plates.

to do

Jennifer Brophy 13:29, 27 February 2009 (EST)

Innoculate two tubes of M10011.
Re-do ligation of M10008 because no colonies grew. Redo plating.

pick M10008 colonies. 
miniprep colonies. Do restriction mapping: (parts >250 bp= EcoRI/BamHI) 
 6.5uL water
 1 uL NEB2
 2 uL plasmid
 0.5 uL EcoRI
 0.5 uL BamHI or XhoI
30 min at 37C 
run gel
turn in for sequencing
 

Jennifer Brophy 01:18, 1 March 2009 (EST)

Picked M10008 colonies. Plate looked very good. Many colonies grew.

to do

Jennifer Brophy 17:08, 1 March 2009 (EST)

Did miniprep of M10008A/B (the two colonies), and M10011A/B.

Gel=ladder, M10008A, M10008B, M10011A, M10011B
Restriction map looks good.

to do

Jennifer Bophy 14:36, 2 March 2009 (EST)

Sent in M10008 and M10011 for sequencing as sb0001, sb0002, sb0003, sb0004.

to do

Jennifer Bophy 14:36, 4 March 2009 (EST)

Analyzed sequencing results of sb0001 (M10008 ad 3 silent point mutations, the part number was changed to M10086) and sb0002 (M10011 was perfect).
Did gateway transfer of Bjb2 {<cpx!} into pBca9495KC.

to do

Jennifer Bophy 13:41, 6 March 2009 (EST)

Miniprep of M10063A and M10063B (two clones of M10063).
Did restriction digest of M10063.
Sent in M10063 for sequencing as sbb052 and sbb053.

to do

Jennifer Bophy 13:23, 11 March 2009 (EDT)

Analyzed the sequence of sbb052: perfect result for M10063.

to do

Jennifer Bophy 14:41, 20 April 2009 (EDT)

Started the Cell-cell adhesion assay. Transformed pBjb1600-Bca1144 (RFP) into DH10B cells. Plated on Spec plates. These cells will eventually be co-transformed with the IILK and AG4 plasmids to test cell density at the end of our experiments.
We are also doing a round of clones without the RFP plasmids, but we did not transform any DH10B cells with plasmids because we are hoping that other groups will have extra colonies.

to do

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Notes

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