Team:Alberta/Building Parts

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(27-05-2010)
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<---gel images of PCR Products (Alina's) and ligated and pre-digested samples (Jeremy's)--->
<---gel images of PCR Products (Alina's) and ligated and pre-digested samples (Jeremy's)--->
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[[User:Anh|Anh]]
 
Digested both A/B' and B/A' Kanamycin Resistance cassettes fragments from [[#11-05-2010|11-05-2010]] and pSB1C3 from [[#20-05-2010|20-05-2010]] with NotI at 37<sup>o</sup>C for 1 hour. Heat inactivated the NotI for 10 minutes at 65<sup>o</sup>C.  Ligated the Kanamycin Resistance cassettes into pSB1C3 at 16<sup>o</sup>C for 1 hour then took 15&mu;L to room temperature for 2 hours.  Transformed 100&mu;L of DH5&alpha; cells with 5&mu;L of RT ligation reaction. Plated transformation on plates with both Chloramphenicol and Kanamycin.
Digested both A/B' and B/A' Kanamycin Resistance cassettes fragments from [[#11-05-2010|11-05-2010]] and pSB1C3 from [[#20-05-2010|20-05-2010]] with NotI at 37<sup>o</sup>C for 1 hour. Heat inactivated the NotI for 10 minutes at 65<sup>o</sup>C.  Ligated the Kanamycin Resistance cassettes into pSB1C3 at 16<sup>o</sup>C for 1 hour then took 15&mu;L to room temperature for 2 hours.  Transformed 100&mu;L of DH5&alpha; cells with 5&mu;L of RT ligation reaction. Plated transformation on plates with both Chloramphenicol and Kanamycin.

Revision as of 17:02, 18 June 2010

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


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 37oC 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 37oC 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
1$mu;L 10 uM dNTPs
2μL 50 uM MgCl2
17.5μL MilliQ H2O
0.5$mu;L 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-94oC
  2. 45 sec-94oC
  3. 1 min-60oC
  4. 1 min-72oC
  5. Repeat 2 through 4 35 times
  6. 5 min-72oC

<---gel images of PCR Products (Alina's) and ligated and pre-digested samples (Jeremy's)--->


Digested both A/B' and B/A' Kanamycin Resistance cassettes fragments from 11-05-2010 and pSB1C3 from 20-05-2010 with NotI at 37oC for 1 hour. Heat inactivated the NotI for 10 minutes at 65oC. Ligated the Kanamycin Resistance cassettes into pSB1C3 at 16oC 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 37oC. 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 37oC for one hour and then with EcoRI at 37oC 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 37oC for 1.5hours, heat inactivated at 65oC 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 16oC.

01-06-2010

KanA/B' and KanB/A' fragements PCRed on 11-05-2010, digested with BsaI-HF at 37oC for 1.5hours, heat inactivated at 65oC 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 21oC.

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 50oC for 1hour, heat inactivated the enzyme at 65oC 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. <--!images-->

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

Anh

Made a 1/100 dilution of AmpR and TetR PCR Products 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-94oC
  2. 45 sec-94oC
  3. 1 min-60oC
  4. 1 min-72oC
  5. Repeat 2 through 4 35 times
  6. 5 min-72oC

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 03-06-2010.

Digestion Recipe:

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
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

Incubated at 50oC for 1hour, heat inactivated the enzyme at 70oC 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 37oC for 1hour, heat inactivated the enzyme at 80oC for 20 minutes.

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

Anh

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

Ligated digested A/B' psB1C3 with AmpR 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 10-06-2010. Hooray~ We made overnights.

12-06-2010

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

16-06-2010

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

<---gel image of Karina's 16.06.10--->

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

Also, we made more overnights of psB1C3 with AmpR.

17-06-2010

Digested psB1C3 with AmpR was ran on a 1% agarose gel.

<---17.06.10 Anh--->


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