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Revision as of 14:24, 9 August 2015
LABJOURNAL
BioSpray
Our forensic toolkit aims to use synthetic biology approaches to improve on the current methods used by crime scene investigators.
Summary
This week was all about getting started with the newly arrived biobrick BBa_K1058008
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To purify BBa_K1058008 for sequence confirmation.
Protocols Used:
Click here to see our miniprep protocol.
Click here to see our sequencing protocol.
Results Miniprep:
|
Sample Name |
Concentration |
Unit |
|
Blank |
- |
- |
|
BBa_K1058008 |
190.93 |
ng/µl |
Results Sequencing: Figure 1
Next Steps: PCR of parts of BBa_K1058008.
Summary
During this week, BBa_K1058008 was disassembled into its constituent parts, pChr, and ChrB.
Aim of experiment: To break down BBa_K1058008 into the promoter region pChr, and the repressor gene, ChrB, while adding standard prefix and suffix to each. Furthermore optimisation of first 15 codons of ChrB in order to increase the overall rate of transcription. Restriction digest of the respective ends and subsequent ligation of pChr and ChrB into pSB1C3 for submission to registry.
Protocols Used:
Click here to see our PCR protocol.
Click here to see our gel extraction protocol.
Click here to see our restriction digest protocol.
Results:
Figure 2: Agarose gel after PCR of pChr and ChrB.
As can be seen on the image of the gel, PCR of ChrB was not successful. pChr was further processed by restriction digest with EcoRI and PstI
Next Steps: Repeat of PCR of ChrB. Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Summary
During this week ligations of pChr and ChrB into pSB1C3 were finalised and the constituent parts of our newly designed chromate sensing system were prepared for ligation and transformation.
Aim of experiment: To repeat PCR of ChrB for subsequent ligation into pSB1C3. Ligation of pChr and ChrB into pSB1C3. Transformation of each into a non-pathogenic lab strain of E. coli.
Overnight culture of strain, containing GFPmut2, as a fluorescent reporter for our Chromate sensing system
Protocols Used:
Click here to see our PCR protocol.
The annealing temperature was lowered in order to increase the chances of accomodating a primer that is not 100% complementary. Furthermore different concentrations of DMSO were tested.
Click here to see our gel extraction protocol.
Click here to see our restriction digest protocol.
Click here to see our ligation protocol. Ligations were made in 2:1 and 3:1 ratio.
Click here to see our Overnight culture protocol.
Results:
Figure 3: Agarose gel after PCR of ChrB.
2 bands were extracted from the gel for further characterisation, the strongest band, hence called ChrBs, and the band directly above, hence called ChrBw.
pChr, ChrBs, and ChrBw were ligated into pSB1C3.
pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 were transformed into E. coli MC1061.
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: Purification of GFPmut2 from overnight cultures. Overnight cultures of recombinant MC1061, containing pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 respectively. Overnight culture of pUniprom for subsequent ligation of ChrB
Protocols Used:
Miniprep for plasmid purification of overnight culture of GFPmut2.
PCR for amplification of GFPmut2 and ChrB.
Gel extraction of PCR product pf GFPmut2.
Overnight culture of MC1061 containing pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 respectively. Furthermore of a strain containing the vector pUniprom.
Results: Figure 5 Since amplification of ChrB was unsuccessful, it will be repeated.
Next Steps: Sequence and size confirmation of pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3.
Aim of experiment: To purify overnight cultures for size and sequence confirmation. Repeat of PCR amplification of pChr and ChrB. Restriction digestes of all parts in order to produce compatible sticky ends.
Protocols Used:
Miniprep of pChr-pSB1C3, ChrBs-pSB1C3, ChrBw-pSB1C3 and pUniprom.
Results Miniprep:
|
Sample Name |
Concentration |
Unit |
|
Blank |
- |
- |
|
pUniprom1 |
55.65 |
ng/µl |
|
pUniprom2 |
64.20 |
ng/µl |
|
pChr1 |
116.58 |
ng/µl |
|
pChr2 |
110.13 |
ng/µl |
|
pChr3 |
114.65 |
ng/µl |
|
pChr4 |
105.63 |
ng/µl |
|
ChrBs1 |
151.18 |
ng/µl |
|
ChrBs2 |
108.37 |
ng/µl |
|
ChrBs3 |
143.13 |
ng/µl |
|
ChrBs4 |
183.27 |
ng/µl |
|
ChrBw1 |
116.26 |
ng/µl |
|
ChrBw2 |
236.99 |
ng/µl |
|
ChrBw3 |
219.21 |
ng/µl |
|
ChrBw4 |
208.27 |
ng/µl |
Protocols Used:
PCR for amplification of pChr and ChrB.
Restriction digest for confirmation of size of pChr and ChrB.
Results: Figure 6 Agarose gel for size confirmation.
Protocols Used:
Sequencing of pChr and ChrB that showed expectes sizes.
Results: Figure 7 Confirmed sequence of pChr1.
Results: Figure 8 Confirmed sequence of ChrBw4.
Protocols Used:
Restriction digest of gel purified PCR-products of pChr, ChrB, and GFPmut2, using EcoRI/SpeI, BamHI/HindIII, and XbaI/PstI respecitvely.
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare pUniprom for insertion of ChrB.
Protocols Used:
Restriction digest of pUniprom with BamHI and HindIII.
Gel extraction of digested pUniprom
Results: Figure 9
Next Steps: Ligation of ChrB into pUniprom.
Aim of experiment: To amplify ChrB version with first 15 codons optimised for insertion into pUniprom.
Protocols Used:
PCR of optimised ChrB from the sequence confirmed ChrBw4-pSB1C3, adding restriction sites compatible to pUniprom.
Gel extraction of optimised ChrBafter PCR.
Results: Figure 10
Next Steps: Ligation of ChrB and codon optimised ChrB into pUniprom.
Aim of experiment: Ligation of ChrB and optimised ChrB into pUniprom. Ligation of pChr and GFPmut2 into pSB1C3
Protocols Used:
Ligation of ChrB and optimised ChrB into pUniprom via BamHI and HindIII restriction sites. Ligation of pChr to GFPmut2 via Spe/Xba link and ligation of pChrGFP construct into pSB1C3 EcoRI/PstI restriction sites.
Results: N/A
Next Steps:Transformation of abovementioned ligations.
Summary
During this week successful transformations were identified and propagated.
Aim of experiment: Transform recombinant vectors into E. coli chassis.
Protocols Used:
Transformation of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3 into JM110.
Results: Figure 11
Aim of experiment: Backup of digested pSB1C3 for the case that a ligation with 2 inserts does not work.
Protocols Used:
Restriction digest of pSB1C3 with EcoRI/SpeI for subsequent insertion of pChr.
Results:N/A
Next Steps: Store digested plasmid at -20oC.
Aim of experiment: Purification of recombinant vectors for sequence and size confirmation.
Protocols Used:
Overnight culture of JM110 with ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3.
Patch plating of the same colonies.
Results: N/A
Next Steps: Purification of the recombinant plasmids for confirmation of size and sequence.
Aim of experiment: Purification of recombinant plasmids for confirmation of size and sequence.
Protocols Used:
Miniprep of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3.
Results:
|
Sample Name |
Concentration |
Unit |
|
Blank |
- |
- |
|
pSB1C3-pChr-GFPmut2_1 |
382.32 |
ng/µl |
|
pSB1C3-pChr-GFPmut2_2 |
355.31 |
ng/µl |
|
pSB1C3-pChr-GFPmut2_3 |
209.85 |
ng/µl |
|
pSB1C3-pChr-GFPmut2_4 |
217.57 |
ng/µl |
|
pUniprom-ChrB-opt_1 |
150.51 |
ng/µl |
|
pUniprom-ChrB-opt_2 |
214.17 |
ng/µl |
|
pUniprom-ChrB-opt_3 |
140.85 |
ng/µl |
|
pUniprom-ChrB-opt_4 |
115.85 |
ng/µl |
|
pUniprom-ChrB_1 |
134.72 |
ng/µl |
|
pUniprom-ChrB_2 |
111.41 |
ng/µl |
|
pUniprom-ChrB_3 |
111.31 |
ng/µl |
|
pUniprom-ChrB_4 |
138.9 |
ng/µl |
Protocols Used:
Restriction digest of ChrB-pUniprom and optimised ChrB-pUniprom with BamHI and HindIII. Restriction digest of pChr-GFPmut2-pSB1C3 with EcoRI/PstI.
Results: Figure 1
Protocols Used:
Colony PCR of pUniprom-ChrB-opt for elucidation of insert size or presence of insert.
Results: Figure 13 Gel after colony PCR. Colonies 2, 4, 9, and 12 were selected for sequencing.
Protocols Used:
Sequencing of pSB1C3-pChr-GFP.
Results: Figure 14 Sequencing result of pSB1C3-pChr-GFPmut2_1. The Result for pSB1C3-pChr-GFPmut2_2 was not as expected, albeit in the correct orientation.
Next Steps: Confirmation of insert size and sequence of ChrB-opt.
Aim of experiment: To prepare ChrB-opt colonies 2, 4, 9, and 12 for sequencing.
Protocols Used:
Click here to see our overnight culture protocol.
Results:N/A
Next Steps: Purification of recombinant plasmids.
Aim of experiment: To purify recombinant plasmids for sequence confirmation.
Protocols Used:
Miniprep of ChrB-opt 2, 4, 9, 12.
Results:
|
Sample Name |
Concentration |
Unit |
|
Blank |
- |
- |
|
pUniprom-ChrB-opt_2 |
255.35 |
ng/µl |
|
pUniprom-ChrB-opt_4 |
146.77 |
ng/µl |
|
pUniprom-ChrB-opt_9 |
211.91 |
ng/µl |
|
pUniprom-ChrB-opt_12 |
181.08 |
ng/µl |
Protocols Used:
Sequencing of ChrB-opt 2, 4, 9, 12.
Results: ChrB showed HindIII restriction sites and was hence cleaved into partial inserts.
Next Steps:Use alternative cloning protocol for ChrB and ChrBopt.
Summary
During this week an alternative cloning strategy for ChrB and ChrB-opt was employed. Furthermore, the pChr-GFP construct was cloned again in a stepwise fashion in order to clone it in the correct orientation.
Aim of experiment:To compare colonies on the patch plate of pSB1C3-pChr-GFP by differentially visualising colonies that do or do not express GFP under UV light.
Results:Figure 15 pSB1C3-pChr-GFP in JM110.
Next Steps:Size confirmation and sequencing of newly selected colonies.
Aim of experiment:Purification of newly selected colonies containing pSB1C3-pChr-GFP for subsequent sequence confirmation.
Protocols Used:
Miniprep for purification of pSB1C3-pChr-GFP.
Results:
|
Sample Name |
Concentration |
Unit |
|
Blank |
- |
- |
|
pSB1C3-pChr-GFP_1 |
313.63 |
ng/µl |
|
pSB1C3-pChr-GFP_5 |
673.70 |
ng/µl |
|
pSB1C3-pChr-GFP_6 |
678.02 |
ng/µl |
|
pSB1C3-pChr-GFP_7 |
537.87 |
ng/µl |
Next Steps:Sequencing of pSB1C3-pChr-GFP samples 1, 5, 6, and 7.
Protocols Used:
Sequencing of pSB1C3-pChr-GFP samples 1, 5, 6, and 7.
Results: Figure 16 Alignment of sequencing results.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Summary
This week was all about getting started with the newly arrived biobrick BBa_K1058008
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Summary
This week was all about getting started with the newly arrived biobrick BBa_K1058008
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Summary
This week was all about getting started with the newly arrived biobrick BBa_K1058008
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Summary
This week was all about getting started with the newly arrived biobrick BBa_K1058008
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Summary
This week was all about getting started with the newly arrived biobrick BBa_K1058008
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.
Protocols Used:
Click here to see our plating protocol.
Click here to see our overnight culture protocol.
Results: N/A
Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.
Restriction digests were set up according to the table below. The appropriate reaction and restriction enzymes that were used is stated in the Journal section.
|
Post Plasmid Purification |
Post PCR |
gBlock |
|
|
DNA |
1mg |
50µl |
100ng |
|
Cutsmart Buffer (µl) |
6 |
6 |
6 |
|
Water (µl) |
As required |
1 |
As required |
|
Enzyme 1 (µl) |
1.5 |
1.5 |
1.5 |
|
Enzyme 2 (µl) |
1.5 |
1.5 |
1.5 |
|
Total Volume (µl) |
30 |
60 |
30 |
The digests were incubated at 37oC for 3 hours.
Note that when performing plasmid digestions, 2.5µl of alkaline phosphatase and 2.5µl of its buffer were added at the 2 hour and 2.5 hour mark.
Ligation reactions were set up according to the table below. The vector to insert ratio is stated for each specific reaction in the Journal section.
|
Vector (µl) |
2:1 (Vector: Insert) [µl] |
3:1 (Vector: Insert) [µl] |
4:1 (Vector: Insert) [µl] |
|
|
T4 DNA Ligase |
1 |
1 |
1 |
1 |
|
Ligase Buffer |
1 |
1 |
1 |
1 |
|
Vector |
1 |
1 |
1 |
1 |
|
Insert |
- |
2 |
3 |
4 |
|
H20 |
7 |
5 |
4 |
3 |
The reactions were incubated at room temperature for at least 3 hours.
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