Team:Alberta/Notebook July

July 1, 2010

Made 5mL liquid cultures with Choramphenicol with streaks from plates of ccdB Bbs A/B', ccdB Bbs B/A', cddB BsaA/B', ccdB BsaB/A' part plasmids from 25-06-2010. Incubated at 37^oC for 1 hour. Plated 25μL of 1/100 and 1/1000 dilutions of liquid cultures onto Chloramphenicol LB plates.

Miniprepped liquid culture of Kan BbsA/B' #1 made 30-06-2010.

Ran gel of Enzyme efficiency experiment performed on 30-06-2010.

Ran gel of digests of Minipreps of ccdB part plasmids performed on 30-06-2010 to ensure they are as expected.

<--AMANDA miniprep of ccdB BfuA/B', ccdB BfuB/A' part plasmids, digest with NotI?-->

July 2, 2010

The colonies on the 1/100 and 1/1000 dilution plates of ccdB Bbs A/B', ccdB Bbs B/A', cddB BsaA/B', ccdB BsaB/A' part plasmids made on 01-07-2010, were spaced appropriately so as to be able to pick individual colonies. We picked as many colonies as possible and streaked on both LB plates with Kanamycin and Chloramphenicol resistance and LB plates with only Choramphenicol resistance. Again to determine if ccdB was infact inserted.

July 6, 2010

Constructing Bsa AB Kan, Chlor and Tet Parts

Set up 5ml overnight tubes with 5ml LB broth, 5ul kanamycin, 2.5ul ampicillin from plates made on 30-06-2010. Results: All seven tubes had growth and no growth in control tube.

Experiment continues on 07-07-2010.

July 7, 2010

Constructing Bsa AB Kan, Chlor and Tet Parts

We made minipreps from the overnights from 06-07-2010. Followed Fermentas protocol to prepare 7 tubes. Restriction Digest with XbaI and PstI Protocol to check for orientation:

Kan AB in pSB1A3 1ul

XbaI enzyme 2ul

PstI enzyme 1ul

NEBuffer 3 4ul

MilliQ H2O 3ul

BSA 4ul

Total 14ul

Incubated at 37C for 1 1/2 hours.

Gel Electrophoresis:

This is a shared gel. Lanes 2-8 are pSB1A3 plasmids with AB Kan inserts from different tubes, digested with XbaI and PstI. The plasmids from lanes 2 and 3 produced proper fragments. The rest of the tubes were discarded.

Restriction Digest with BsaI Protocol:

Kan AB in pSB1A3 (247.4 ng/ul or 255.8 ng/ul) 1ul

chlor or tet AB fragment 1ul

BsaI enzyme 0.5ul

NEBuffer 4 3ul

MilliQ H2O 5.5ul

Total 11ul

Incubated at 50C for 1 hour.

Ligation Protocol:

Digested pSB1A3 with Kan AB and chlor or tet 8ul

T4 DNA ligase 1ul

5X T4 DNA ligase buffer 6ul

MilliQ H20 5ul

Total 20ul

Incubated at room temperature for 1 hour.

Gel Electrophoresis:

Experiment continues on 08-07-2010.

July 8, 2010

Constructing Bsa AB Kan, Chlor and Tet Parts

Followed Inoue transformation protocol using 50ul DH5-alpha competent cells and 2ul of pSB1A3 ligated with tet or chlor made on 07-07-2010. Plated 200ul and 600ul on chlor/amp plates for chlor insert and on tet/amp plates for tet insert. Results:

chlor/amp plate 1 (200ul): ~300 colonies

chlor/amp plate 2 (600ul): TNTC colonies

tet/amp plate 1 (200ul): ~20 colonies

tet/amp plate 2 (600ul): ~30 colonies

Experiment continues on 09-07-2010.

July 9, 2010

Constructing Bsa AB Kan, Chlor and Tet Parts

Made overnights with 5ml LB, [20ul chlor and 2.5 ul amp] or [20ul tet and 2.5 ul amp] using transformation plates from 08-07-2010. Results were growth in all experimental tubes and no growth in control tubes.

Experiment continues on 10-07-2010.

July 10, 2010

Constructing Bsa AB Kan, Chlor and Tet Parts

We prepared minipreps of tet in pSB1A3 (4 tubes) and chlor in pSB1A3 (7 tubes) from overnights 09-07-2010.

Experiment continues on 12-07-2010.

July 12, 2010

Constructing Bsa AB Kan, Chlor and Tet Parts

Restriction Digest with EcoRI Protocol to determine plasmid size:

pSB1A3 with chlor or tet insert 1ul (made on 10-07-2010)

Fast Digest EcoRI 1ul

10X Fast Digest Buffer 2ul

MilliQ H2O 16ul

Total 20ul

Incubated at 37C for 5 mins.

Gel Electrophoresis:

Lanes 2-5 are pSB1A3 plasmid with tet AB insert. Lanes 6-13 are pSB1A3 plasmid with chlor AB insert. All plasmids were digested with EcoRI. All but the products of lanes 3,6,10,11,12 were correct. 1 out of the 4 tet in pSB1A3 tubes cut incorrectly and was discarded, 4 out of the 7 chlor in pSB1A3 tubes cut incorrectly and was discarded.

Experiment continues on 14-07-2010.

July 14, 2010

Constructing Bsa AB Kan, Chlor and Tet Parts

Double Digest of chlor, kan and tet in PSB1A3 and amp is pSB1C3 with Xba and PstI protocol:

plasmid with antibiotic part 1ul

10X NEBuffer 3 1ul

MilliQ H20 5ul

XbaI 1ul

PstI 1ul

10X BSA 1ul

Total 10ul

Incubated at 37C for 1 hour.

Gel Electrophoresis:

Digests of antibiotic AB fragments with XbaI and PstI. Lanes 2-3 are kanamycin AB, lanes 4-6 are chloramphenicol AB, lanes 7-9 are tetracycline AB, lanes 10-12 are ampicillin AB. All of the plasmids cut correctly except for one of the amp in pSB1C3, which was discarded.

Experiment continues on 15-07-2010.

July 15, 2010

Constructing Bsa AB Kan, Chlor and Tet Parts

Antibiotic parts, made on 14-07-2010, are now stored in the DNA box according to the BioBytes registry.

Glycerol stocks were made of overnights from chlor, tet, and kan in PSB1A3 plates.

July 19, 2010

Constructing a new plasmid with Bfu AB or BA Kan in pSB1A3

Digest of pSB1A3 and premade Bfu AB kan fragments with NotI protocol:

pSB1A3 (30.5ng/ul) 3ul

10X REact 3 Buffer 1ul

MilliQ H20 10ul

Bfu Kan AB (38.1ng/ul) 3ul

NotI 1ul

10X BSA 2ul

Total 20ul

Incubated at 37C for 1 hour.

Digest of pSB1A3 and premade Bfu BA kan fragments with NotI protocol:

pSB1A3 (30.5ng/ul) 3ul

10X REact 3 Buffer 1ul

MilliQ H20 10ul

Bfu Kan BA 2ul

NotI 2ul

10X BSA 2ul

Total 20ul

Incubated at 37C for 1 hour.

Ligation of pSB1A3 and premade Bfu AB/BA kan fragments:

Digests from above 10ul

5X Ligase Buffer 4ul

MilliQ H20 5ul

T4 DNA ligase 1ul

Total 20ul

July 20, 2010

Constructing a new plasmid with Bfu AB or BA Kan in pSB1A3

Followed Inoue transformation protocol using 50ul DH5-alpha competent cells and 2ul of pSB1A3 ligated with kan AB or BA. Plated on kan/amp plates.

July 21, 2010

Constructing a new plasmid with Bfu AB or BA Kan in pSB1A3

kan AB 1: 3 white colonies

kan AB 2: 50 white, 1 red colonies

kan AB 3: 45 white, 2 red colonies

kan BA 1: 25 white colonies

kan BA 2: 17 white, 1 red colonies

kan BA 3: 25 white, 1 red colonies

Set up overnights with 5 ul kan and 2.5 ul amp per 5ml LB

July 22, 2010

Constructing a new plasmid with Bfu AB or BA Kan in pSB1A3

Miniprepped overnight tubes (growth in all).

Digest of pSB1A3 with Bfu kan AB/BA inserts with XbaI and PstI enzymes protocol:

plasmid DNA ul

10X NEBuffer 3 2ul

MilliQ H20 12ul

XbaI 1ul

Pst I 1ul

10X BSA 2ul

Total 20ul

Incubated for 1 hour at 37C.

July 23, 2010

Constructing Bfu AB/BA chlor, amp and tet parts

Digest of Bfu Kan AB/BA in pSB1A3 and pSB1C3 with BfuAI protocol:

psb1A3 with Bfu Kan AB/BA insert OR psb1C3 with Bfu Kan AB/BA insert 5ul

BfuAI 1ul

NEBuffer 3 10ul

MilliQ H20 84ul

Total 100ul

Incubated at 50C for 1 hour. Heat inactivated at 65C for 20 minutes.

Insert gel here

Digest of Bsa AB chlor, tet and amp in PSB1C3 with BsaI protocol:

psb1C3 with Bsa AB chlor, tet and amp 5ul

BsaI 1ul

NEBuffer 4 10ul

MilliQ H20 84ul

Total 100ul

Incubated at 50C for 1 hour. Heat inactivated at 65C for 20 minutes.

Insert gel here

Ligations of Bfu amp AB/BA into pSB1C3 and Bfu chlor and tet AB/BA into pSB1A3 protocol:

pSB1A3 or pSB1C3 10ul

antibiotic insert 10ul

5x ligase buffer 8ul

T4 DNA ligase 1ul

Total 40ul

Incubate at room temperature for one hour.

July 26, 2010

Constructing Bfu AB/BA chlor, amp and tet parts

Followed Inoue transformation protocol using 50ul DH5-alpha competent cells and 2ul of pSB1A3 or pSB1C3 ligated with AB and BA antibiotics. Plated on various antibiotic plates.

July 28, 2010

Constructing Bfu AB/BA chlor, amp and tet parts

Overnights Results

Growth: Bfu AB amp1,2,3; Bfu AB chlor1,2,3; Bfu BA chlor1,2

No growth: Bfu AB chlor2, Bfu BA chlor3

Tubes with growth were miniprepped.