Team:Alberta/Building Parts
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:
- 5 min-94oC
- 45 sec-94oC
- 1 min-60oC
- 1 min-72oC
- Repeat 2 through 4 35 times
- 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:
- 5 min-94oC
- 45 sec-94oC
- 1 min-60oC
- 1 min-72oC
- Repeat 2 through 4 35 times
- 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
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:
- 5 min-94oC
- 45 sec-94oC
- 1 min-60oC
- 1 min-72oC
- Repeat 2 through 4 35 times
- 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 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 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--->
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 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.
<---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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