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From 2012.igem.org

Hyder's lab notes

6/22/12

miniprepped gfp1 and rbs1 and rbs2 liquid cultures

picked 1 colony from double terminator (dt1) plate

picked 1 colony from t7 polymerase (pol1) plate

picked 1 colony from puc19 plate (positive control)

picked 1 colony from dh5a plate (negative control)

started liquid cultures of each colony (5 mL LB amp each)

8/3/12

Resuspended GFPT1 and GFPT2 oligos with molecular grade (nuclease-free) H2O.

Final Concentration 100uM

(gfpt1 top1, gfpt2 top1, gfpt1 top2, gftp2 top2, gfpt1 bot1, gfpt2 bot1, gfpt1 bot2, gfpt2 bot2)

(3uL of each oligo + 2uL 10x annealing buffer, 6uL molecular grade H2O. 20uL Reactions)

Heated for 5 minutes at 100C. Let cool to room temperature on the heating block, stored at -20C.

Digested BBa_I13522 with XbaI and PstI.

Attempted ligating annealed oligos into a digested ?kill plasmid? from Ryan (realized it was cut with E and P).

8/6/12

Annealed oligos for GFPT1 and GFPT2 (target probes)

Ligated oligos with digested GFP plasmid (BBa_I13522)

Transformed into competent DH5alpha

Added SOC and incubated at 37C for 15 minutes.

Plated on amp treated plates.

8/7/12

Only one colony on each plate (both were white)

Picked colonies and started 5mL LB amp cultures of each, stored at 37C

Stored plates in 37C

8/8/12

Picked the colonies again and started new liquid cultures (5mL LB amp).

Discarded cultures for 8/7/12

8/9/12

Miniprepped 3mL of each 8/8/12 culture and nanodropped:

gfpt1 - 155 ng/uL

gfpt2 - 114 ng/uL

Digested gfpt1 and gfpt2 with X and P

Ran on a 1% agarose gel with the digested GFP plasmid

Made glyercol stocks with aliquot of the remaining liquid cultures

8/13/12

Prepared sequencing samples

• Sample w/ Primer*

GFPT1 w/ VF2 GFPT1 w/ VR GFPT2 w/ VF2 GFPT2 w/ VR

200ng of DNA + 16 pmol of primer

Annealed oligos again GFPT1/2

Repeated ligation of oligos with digested GFP plasmid (BBa_I13522)

Followed Haynes assembly protocol instead of standard DH5alpha protocol. (http://openwetware.org/wiki/Haynes:Assembly101)

Transformed ligations into competent DH5alpha

Plated on amp treated plates

8/14/12

Took pictures of plates

Green-white screened plates

Picked 4 white colonies from each of gfpt1/2 plates

Made 5mL LB amp cultures of each colony

Delivered GFPT1/2 dna samples to biodesign for sequencing (samples from 8/13/12)

8/15/12

Miniprepped 3mL of each liquid culture of GFPT1/2

Prepared glycerol stocks using 100uL of each liquid culture

Nanodropped samples:

GFPT1-1 - 172.6 ng/uL GFPT1-2 - 203.7 ng/uL GFPT1-3 - 197.4 ng/uL GFPT1-4 - 178.9 ng/uL GFPT2-1 - 107.3 ng/uL GFPT2-2 - 131.2 ng/uL GFPT2-3 - 145.5 ng/uL GFPT2-4 - 172.0 ng/uL

8/17/12

GFPT1 sequence confirmed

Prepared aliquots of GFPT2 minipreps from 8/15/12 for sequencing

Delivered GFPT2 samples to biodesign for sequencing

8/19/12

GFPT2 sequences confirmed

8/27/12

Revived GFPT1 (from 8/9/12) and GFPT2 (2-2 from 8/15/12) cultures from glycerol scrapes

Made 4mL cultures in LB Amp

8/29/12

Discarded GFPT1/2 cultures from 8/27/12

Revived GFPT1 (from 8/9/12) and GFPT2 (2-2 from 8/15/12) cultures from glycerol scrapes

Made 4mL cultures in LB Amp

Digested GFPT1/2 with X and S

Ran a 1% agarose gel with GFPT1/2 digestions

Cut out inserts and GFPT2 backbone and stored in 4C for gel extraction and tandum repeat assembly experiments

8/30/12

Prepared extra glyercol stocks of GFPT1/2 cultures from 8/29/12

Miniprepped 3mL of each culture, stored at -20C

9/19/12

Set up VF2/VR endpoint PCR for double transform minipreps

1-1, 1-1I, 1-2, 1-2I, 1-3, 1-3I, 2-1, 2-1I, 2-2, 2-2I, 2-3, 2-3I, GFPT1 (positive controls), GFPT2 (positive controls)

Annealing temp set to 55C for 25 cycles

Resuspended GFPT1 probe and GFPT2 probe oligos in molecular grade H2O (Final concentration: 100uM), stored at -20C

9/25/12

Resuspended pSB1A2 FWD and pSB1A2 REV (amp resistance primers) oligos in molecular grade H2O (Final concentration: 100uM), stored at -20C

Prepared 1.6uM dilutions (500uL)

Did endpoint PCR using pSB1A2 primer pair on

Topo, Topo IPTG, Topo D168A, Topo D168A IPTG, 1-1, 1-1I, 2-1, 2-1I, GFPT1, GFPT2

Did endpoint PCR using VF2/VR primer pair on

Topo, Topo IPTG, Topo D168A, Topo D168A IPTG

Annealing temp 55C for 25 cycles, stored products at -20C

Made 3mL liquid cultures of the shipping vector (pSB1C3 with RFP insert) in DH5alpha in chloramphenicol resistant LB

Made 10mL liquid cultures of:

Topo in kanamycin

topo D168A in kanamycin

topo + GFPT1 in kanamycin + ampicillin

topo + GFPT2 in kanamycin + ampicillin

stored @ 37C

9/26/12

Picked two new colonies for topo + GFPT2 and grew 10mL cultures of amp+kan LB broth for each

Ran a gel with:

Hyperladder I, GFPT1 pSB1A2, GFPT2 pSB1A2, Topo pSB1A2, Topo D168A

Ran a gel with PCR samples from 9/19 and 9/25:

Hyperladder I, 1-1, 1-1I, 2-1, 2-1I, 2-1I(VF), 2-1(VF), 1-1I(VF), 1-1(VF), Hyperladder I

Samples in wells 2-5 used the pSB1A2 primer pair. Samples in wells 6-9 used VF2/VR primer pair.

Revived GFPT1/2 from glycerol stocks (from 8/9 and 8/15) in 5mL LB amp each.

Nanodropped miniprepped DNA samples:

pSB1C3 I - 253.75 ng/uL

pSB1C3 II - 258.38 ng/uL

Topo - 24.84 ng/uL

Topo I - 17.33 ng/uL

Topo D168A - 7.24 ng/uL

Topo D168A I - 15.62 ng/uL

1-1 - 16.49 ng/uL

1-1I - 16.46 ng/uL

2-1 - 142.05 ng/uL

2-1I - 16.98 ng/uL

Picked new colonies from Topo, Topo D168A, Topo + G1, Topo + G2 and made 1mL colonies in their respective medias

Stored at -37C

9/27/12

Prepared serial dilutions of GFPT2 plasmid 1:10, 1:100, 1:1000, 1:10000

Prepared primer mixes for pSB and VF/VR primers

Prepared realtime PCR plate, ran RT-qPCR with annealing temp 57

Miniprepped GFPT1/2 cultures from 9/26

Nanodropped Miniprepped DNA:

GFPT1 - 276.12 ng/uL

GFPT2 - 219.84 ng/uL

Used 100uL of each 1mL culture from 9/26 to seed 10 mL cultures in their respective media

Added 10uL of 1M IPTG to each culture ~4 hours after seeding

Removed cells from 37C ~4 hours after IPTG inducing

Pelleted and lysed following the bugbuster protocol (http://openwetware.org/wiki/User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2012/05/22) (used lysonase for Topo and Topo D168A only)

HIS purified proteins using the Zymo HIS purification kit

Digested pSB1C3 plasmid with X and P

Sent protein samples (HIS purified samples from 9/19, 20uL) and ssDNA control (GFPT1/2 probe oligos 20uL @ 10uM) for mass spec

9/28/12

Resuspended 8 new oligos, final volume 100 uM each

Annealed pet29 top/bot oligos

Redid the RT-PCR, 3x primer concentration, added 1:1 plasmid concentration

Made 1.6uM aliquots of primer stocks 1-7 (including topo add X primer previously ordered)

Diluted aliquot of Topo D168A plasmid 4:10

Diluted PSV plasmid 2:20

Performed Endpoint PCR on:

Topo D168A

using primers 1,2 and primers 1,3

PSV using primers 4-5 and primers 6-7

4 samples with low primer concentration, 4 samples with twice as much primer (labelled 'H')

Miniprepped Topo1 2, Topo1 3, Topo1 4 (biobricked Topo D168A without T7, multiple colonies from ligation into shipping vector)

Nanodropped:

Topo1 2 - 63.63 ng/uL

Topo1 3 - 75.07 ng/uL

Topo1 4 - 184.93 ng/uL

1mL chloramphenicol cultures of GFPT1/GFPT2 in shipping vector prepared

Digested Topo1 2, Topo1 3, Topo1 4 with E and P

Ran digested Topo plasmids on 1% agarose gel with Hyperladder I

Revived cultures of 2xGFPT1, 2xGFPT2, Topo, and Topo D168A (4 mL cultures each in their respective medias)

T5 exonuclease treated miniprepped plasmids (1-1, 1-1I)

A1 1-1I + t5

B1 1-1 + t5

A2 1-1I untreated

B2 1-1 untreated

9/29/12

Miniprepped GFPT1, GFPT2, Topo, and Topo D168A (Topo cultures separated into 3mL and 1mL minipreps)

Nanodropped miniprepped DNA:

GFPT1 I - 275.29 ng/uL

GFPT1 II - 101.08 ng/uL

GFPT2 I - 222.11 ng/uL

GFPT2 II - 230.52 ng/uL

Topo I - 117.97 ng/uL

Topo II - 70.28 ng/uL

Topo D168A I - 69.47 ng/uL

Topo D168A II - 51.87 ng/uL

HIS purified crude lysates from 9/27/12 (Topo, Topo D168A, Topo + GFPT1, Topo + GFPT2, all IPTG induced)

Digested Topo I plasmid (pet29a) with E and X

Ran a gel with Hyperladder I, 1-2 1-3 4-5 and 6-7 PCR products, pet29a digestion, and pSB1C3 digestion

Confirmed that PCR made amplicons

Excised bands for digested pet29a and pSB1C3 plasmids

Gel extracted 4 gel fragments (2 wells per sample: digested pSB1C3 plasmid, digested pet29a plasmid)

Nanodropped gel extractions:

Digested pSB1C3 - 25.54 ng/uL

Digested pet29a - 22.95 ng/uL

Set up a bradford assay of topo, topo D168A, topo + G1, topo + G2 (5uL protein, 10uL protein, 20uL protein + 200uL reagent)

Used ~100uL aliquot of BL21 competent glycerol stock to seed 10mL of LB medium (no antibiotic), stored at 37C

Ran 1% agarose gel with samples: A1, B1, A2, B2 and Hyperladder I

Did bug buster protocol to lyse BL21 control culture (used lysonase)

Ligated digested pet29a with pet29 top/bot annealed oligos

Transformed ligation using invitrogen DH5alpha transformation protocol

Prepared LB kanamycin plates

plated transformed ligations on prewarmed kanamycin plates

9/30/12

Pet29a plates did not grow

Kinase treated pet29a oligos

Annealed kinase treated pet29a oligos

Ligated digested pet29a (from gel extraction) with kinase treated oligos

Transformed ligations into DH5alpha, used topo plasmid as a positive control

Plated transformations on kanamycin plates and stored overnight at 37C

HIS purified BL21 control crude lysate

Set up a bradford assay with:

uninduced & induced topo protein extractions from 9/19

uninduced & induced topo D168A protein extractions from 9/19

BL21 control lysate

Topo, Topo D168A, Topo + G1, Topo + G2 from 9/27

All samples prepared (10uL protein, 20uL protein + 200uL reagent)

Miniprepped GFPT1 1,2,3 and GFPT2 (17,18,26) (~600uL of each) (1mL liquid cultures made from colonies on the chloramphenicol plates of GFPT1 and GFPT2 ligated into the shipping vector)

Nanodropped:

GFPT1-1 - 38.5 ng/uL

GFPT1-2 - 77.6 ng/uL

GFPT1-3 - 74.8 ng/uL

GFPT2-17 - 61.6 ng/uL

GFPT2-18 - 65.9 ng/uL

GFPT2-26 - 64.2 ng/uL

Ran a 1% agarose gel with 1-1, 1-2, 1-3, 2-17, 2-18, 2-26 plasmid miniprep samples and Hyperladder I

Prepared a 1:2 dilution of GFPT2 plasmid from 9/27 miniprep

Treated 5uL of diluted plasmid with 5uL of water, BL21 protein, topo protein, topo D168A protein

Incubated 30 minutes at 37C

Ran a 1% agarose gel with protein treated target plasmid samples and Hyperladder I

Digested GFPT2 plasmid with X (let run at 37C for 30 minutes)

Used DNA clean up kit on digested GFPT2

Nanodropped digested GFPT2:

GFPT2(X) - 39.85 ng/uL

Prepared DNA seq samples using VF2 and VR

Sample# - PrimerPair - DNA sample (sample 1, GFPT2 uncut + VF2; sample 2, GFPT2 uncut + VR)

1/2 - FWD/REV - uncut GFPT2 plasmid

3/4 - FWD/REV - cut GFPT2 plamid

5/6 - FWD/REV - 2:1 uncut:cut GFPT2 plasmid mixture

7/8 - FWD/REV - 1:1 uncut:cut GFPT2 plasmid mixture

9/10 - FWD/REV - 1:2 uncut:cut GFPT2 plasmid mixture

11/12 - FWD/REV - 1-2 miniprep sample from 9/19 double transformations

13/14 - FWD/REV - 1-2I

15/16 - FWD/REV - 1-3

17/18 - FWD/REV - 1-3I

19/20 - FWD/REV - 2-1

21/22 - FWD/REV - 2-1I

23/24 - FWD/REV - 2-2

25/26 - FWD/REV - 2-2I

27/28 - FWD/REV - 2-3

29/30 - FWD/REV - 2-3I

10/2/12

Prepared PCR tubes with 10uL Topo1 4 (25ng/uL topo d168a in the shipping vector), 10uL GFPT2-26 (25ng/uL GFPT2 in the shipping vector), and 10uL GFPT1-3 (25ng/uL GFPT1 in the shipping vector)

Labelled the tubes K891234, K891999, K891000 respectively and shipped overnight to iGEM Headquarters