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From 2010.igem.org

Building Parts

10-05-2010

PCR a Kanamycin resistance cassette fragment from p1003 with primers containing either the A/B' ends or the B/A' ends.

Recipe:

1μL p1003 (approx. 1ng)

2.5μL prA_p1003+

2.5μL prB'_p1003-

5μL 10X PCR buffer

1μL 10uM dNTPs

2μL 50uM MgCl₂

0.5μL Taq polymerase

35.5μL MilliQ H₂O

Program:

1. 5 min-94^oC
2. 45 sec-94^oC
3. 1 min-60^oC
4. 1 min-72^oC
5. Repeat 2 through 4 35 times
6. 5 min-72^oC

11-05-2010

PCR purification of Kanamycin Resistant fragments created 10-05-2010 with Qiagen PCR cleanup kit. Determined concentrations by nanodrop. KanA/B': 101.1ng/μL KanB/A':89.6ng/μL

18-05-2010

Prepared DH5α E.Coli competent cells using the Inoue Method. Transformed DH5α cells with pSB1C3-J04450 and grew overnight at 37^oC on Chloramphenicol plates

19-05-2010

From the transformation of DH5α cells with pSB1C3-J04450 performed on 18-05-2010, we took 4 distinct colonies, streaked them on a new chloramphenicol plate and inoculated 5ml liquid cultures.

20-05-2010

Performed a Miniprep of 3 of the 4 5ml liquid cultures of DH5α cells with pSB1C3-J04450 from 19-05-2010. Took a 1μL sample of the Miniprep solutions and digested with NotI at 37^oC for 1 hour.

Digestion Recipe:

1μL Miniprep (between 153.2 ng/μl and 302.7ng/μl determined by nanodrop)

1μL NotI

1μL 10X ReACT 3

7μL MilliQ

27-05-2010

Performed PCR reactions to create parts with antibiotic resistance with negative controls.

PCR Recipe:

3μL 10X PCR Buffer

1μL 10 uM dNTPs

2μL 50 uM MgCl₂

17.5μL MilliQ H₂O

0.5μL Taq Polymerase

1μL Template (psB4A5-J04450, psB4C5-J04450 or psB3T5-J04450)

2.5μL Primer + (PrA psB4A5 ApR+, PrA psB4C5 ChR+ or PrA psB3T5 TR+)

2.5μL Primer - (PrB psB4A5 ApR-, PrB psB4C5 ChR- or PrB psB3T5 TR-)

PCR Program:

1. 5 min-94^oC
2. 45 sec-94^oC
3. 1 min-60^oC
4. 1 min-72^oC
5. Repeat 2 through 4 35 times
6. 5 min-72^oC

Digested both A/B' and B/A' Kanamycin Resistance cassettes fragments from 11-05-2010 and pSB1C3 from 20-05-2010 with NotI at 37^oC for 1 hour. Heat inactivated the NotI for 10 minutes at 65^oC. Ligated the Kanamycin Resistance cassettes into pSB1C3 at 16^oC for 1 hour then took 15μL to room temperature for 2 hours. Transformed 100μL of DH5α cells with 5μL of RT ligation reaction. Plated transformation on plates with both Chloramphenicol and Kanamycin.

Digestion Recipe:

1μL Miniprep (302.7ng/μl determined by nanodrop)

2μL either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/μL)

1μL NotI

1μL 10X ReACT 3

5μL MilliQ

Ligation Recipe:

10μL of Digest solution

1μL T4 DNA ligase

6μL 5X Buffer

13μL MilliQ H₂O

Also transformed pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450 from the 2010 biobrick parts into DH5α cells.

28-05-2010

We got colonies!! (it's a fantastic feeling) We then lovingly put them in the cold room to await our return from Calgary

30-05-2010

From the transformation of DH5α cells with pSB1C3-KanR performed on 28-05-2010, we took 12 distinct colonies of each KanA/B' and KanB/A', streaked them on a new chloramphenicol plate and inoculated 5ml liquid cultures with the appropriate antibiotics overnight at 37^oC. We also picked colonies of pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450, streaked and made 5mL liquid cultures of them too.

31-05-2010

9/12 of the pSB1C3-KanA/B' Liquid cultures 30-05-2010 were successful and only 1/12 of the pSB1C3-KanB/A' liquid cultures were successful. The pSB4A5, pSB3T5 and pSB4C5 transformations worked. Miniprepped all the liquid cultures that worked.

Performed a restriction digest of an aliquot of the pSB1C3-KanA/B' and pSB1C3-KanB/A' minipreps. Digested with XbaI at 37^oC for one hour and then with EcoRI at 37^oC for one hour. Ran a 1% agarose gel of the digests to determine the orientation of the KanR fragments.

KanA/B' and KanB/A' fragements PCRed on 11-05-2010, digested with BsaI at 37^oC for 1.5hours, heat inactivated at 65^oC for 30 minutes. Tried to ligate KanA/B' fragments to each other and tried to ligate KanB/A' fragments to each other. Also tried to ligate KanA/B' fragments with KanB/A'. Ligated with T4 DNA ligase overnight at 16^oC.

01-06-2010

KanA/B' and KanB/A' fragements PCRed on 11-05-2010, digested with BsaI-HF at 37^oC for 1.5hours, heat inactivated at 65^oC for 30 minutes. Tried to ligate KanA/B' fragments to each other and tried to ligate KanB/A' fragments to each other. Also tried to ligate KanA/B' fragments with KanB/A'. Ligated with T4 DNA ligase for 3 hours at 21^oC.

Set up liquid cultures from plates streaked on 30-05-2010

02-06-2010

Miniprepped liquid cultures from 01-06-2010. Ran a 1% agarose gel of the ligations performed 01-06-2010. To optimize the Restriction and ligation of BsaI-HF, digested KanA/B' and KanB/A' fragements PCRed on 11-05-2010 with the following recipe:

Digestion Recipe:

14μL either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/μL)

5μL 1/10 dillution of 100X BSA

5μL 10X NEBuffer4

1.5&mu:L BsaI-HF

24.5μL MilliQ

Digested at 50^oC for 1hour, heat inactivated the enzyme at 65^oC for 20 minutes PCR purified the digests.

03-06-2010

Tried to ligate the KanA/B' fragments to itself. Tried to ligate the KanB/A' fragments to itself. Tried to ligate the KanA/B' fragments to the KanB/A' fragments.

Ligation Recipe:

8μL of digest from 02-06-2010 (either 8μL of one of the fragments or 4μL of each)

1μL T4 DNA ligase

6μL 5X Buffer

15μL MilliQ H₂O

Took aliquots of ligations at varying times and ran 1% agarose gels to test ligation.

Digested some of Minipreps of the KanA/B' fragment inserted into pSB1C3 from 02-06-2010. Digestion Recipe:

14μL plasmid (approx 300-400ng/&mu:L)

5μL 10X NEBuffer 4

5μL 1/10 dilution of 100X BSA

1.5μL BsaI-HF

24.5μL MilliQ H₂O

Made a 1/100 dilution of AmpR and TetR PCR Products from 27-05-2010 and performed PCR reactions to produce antibiotic inserts to make parts.

PCR Recipe:

35.5μL MilliQ H₂O

1μL 10 uM dNTPs

5μL 10X PCR Buffer

2μL 50 uM MgCl₂

2.5μL Primer + (ApR 1/10+ or TR 1/10+)

2.5μL Primer - (ApR 1/10- or TR 1/10-)

1μL Template (1/100 diluted AmpR or TetR)

0.5μL Taq Polymerase

PCR Program:

1. 5 min-94^oC
2. 45 sec-94^oC
3. 1 min-60^oC
4. 1 min-72^oC
5. Repeat 2 through 4 35 times
6. 5 min-72^oC

04-06-2010

Tried to test the limits of ligation reaction Ligation Recipe of plasmids cut on 03-06-2010:

8μL digestion mixture

6μL 5X T4 ligase buffer

1μL T4 ligase

15μL MilliQ

Tried to further reaction of KanA/B' fragments to KanB/A' fragments. To the existing Ligase reactions from 03-06-2010 added:

1μL T4 ligase

6μL 5X T4 ligase buffer

23μL MilliQ

Tried to set limits of Kan fragments that would ligate.

24μL digestion from 02-06-2010 (either 24μL of one of the fragments or 12μL of each)

6μL 5X T4 ligase buffer

1μL T4 ligase

09-06-2010

Restriction Digested AmpR and TetR inserts from 03-06-2010 to be ligated with psB1C3 vector later on.

Digestion Recipe for AmpR:

13.4μL MilliQ H₂O

0.60&mu:L AmpR (333.3 ng/μL, determined by nanodrop)

2μL 10X BSA

2μL 10X Buffer 4

2μL BsaI

Digestion Recipe fro TetR:

13.3μL MilliQ H₂O

0.7&mu:L TetR (571.3 ng/μL, determined by nanodrop)

2μL 10X BSA

2μL 10X Buffer 4

2μL BsaI

Both digestions were incubated at 50^oC for 1hour, heat inactivated the enzyme at 70^oC for 20 minutes.

10-06-2010

Double Digested Kan/Chlor minipreps <---Find date of Kan/Chlor production---> to determine orientation.

Digestion Recipe:

5μL 10X Buffer 3

1μL XbaI

1μL PstI

5μL Kan/Chlor fragments

33μL MilliQ H₂O

5μL 10X BSA

Incubated at 37^oC for 1hour, heat inactivated the enzyme at 80^oC for 20 minutes.

<---gel image of 10.06.10 Karina1--->

Restriction Digested A/B' psB1C3 vector.

Digestion Recipe:

10μL MilliQ H₂O

5&mu:L psB1C3

2μL 10X BSA

2μL 10X Buffer 4

1μL BsaHF

Incubated at 37^oC for 1hour, heat inactivated the enzyme at 80^oC for 20 minutes.

Ligated digested A/B' psB1C3 with AmpR from 09-06-2010.

Ligation Recipe:

9μL A/B' psB1C3 vector

5μL AmpR insert

6μL 5X Ligase Buffer

1μL Ligase

9μL MilliQ H₂O

Incubated at room temperature for 45 minutes. Transformed with DH5α cells using 15μL of the ligated A/B' psB1C3 with AmpR.

11-06-2010

We got colonies of psB1C3 with AmpR from the DH5α transformations from 10-06-2010. Hooray~ We made overnights.

12-06-2010

Miniprep of psB1C3 with AmpR was made from the overnights from 11-06-2010.

16-06-2010

psB1C3 with AmpR miniprep from 12-06-2010 ran undigested on a 1% agarose gel.

We digested psB1C3 with AmpR to see if the AmpR insert would be released from the psB1C3 vector.

Digestion Recipe:

14.1μL MilliQ H₂O

0.9μL psB1C3 with AmpR (222.5 ng/μL, determined by nanodrop)

2μL 10X BSA

2μL 10X Buffer 4

1μL BsaI

Incubated at 50^oC for 1hour, heat inactivated the enzyme at 65^oC for 30 minutes.

Also, we made more overnights of psB1C3 with AmpR.

17-06-2010

Digested psB1C3 with AmpR from 16-06-2010 was ran on a 1% agarose gel.

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