Team:GDSYZX/Notebook
Experimental record
7.20
Make LB culture medium, TBE, EDTA
7.21
Commence the PCR of ugt33 target fragment.
7.22
Perform agarose gel electrophoresis test, gel recycling and purification. (ugt33)
7.23
Morning: Purpose piece recycling. (ugt33)
Afternoon: Enzyme digestion. (ugt33)
7.24 (ugt33)
Electrophoresis test, re-purification of DNA solution
No positive clones were detected by the enzyme, and the enzyme may not be cleaned because of the poor quality of the enzyme.
7.25 (ugt33)
DNA conversion.
Plate culture.
7.26 (ugt33)
Colony PCR did not get positive results, and the sequence was wrong
Plasmid transformation
7.27 (ugt33)
Re-heat-induced transformation, plating, re-enzyme.
2019.7.28 (ugt33)
1. Today's electrophoresis (colon PCR, PCR purification product to marker) did not show a band, after re-changing the dye, we re-run electrophoresis and found a band. After analysis, it may be considered that there is a problem with the SYBR Green dye in the small tube, and the dye should be replaced on the second day.
2. Really re-do the transformation of UGT33 gene ligation product
2019.7.29. (ugt33)
1. Replaced the nucleic acid dye, the two teams repeated yesterday's colony PCR experiment
2. Extracted the plasmid containing the positive colonies of RrUGT33. The plasmid concentration is generally about 200 ng/ul. Has been sent for testing. (result is negative)
3. Really carry out the colony PCR on the transformed strain plate yesterday, there is a positive band, and 4 shakes have been selected.
2019.7.30 plan
waiting for the sequencing results, and another colony PCR after the transformation of the recombinant product (If Miao teacher can do the conversion tonight)
2019.7.30. Summary
1. after extracting plasmid of RrUGT33 of PUC-HA, conduct enzyme digestion verification, seven are background, not connected.
2. Amplification of RrUGT33, Rr4HPAAS, no banding in electrophoresis, after analysis, it is considered that there may be problems with Q5 enzyme activity or other reagents.
2019.7.31. Tomorrow's plan
1. Reconstitute 5xTBE
2. Imidazole and Na2HPO4 are required to prepare a loading buffer.
3. A 0.22 um filter is required and the PBS buffer is filtered.
4. Purchase SDS-PAGE gel preparation kit.
5. If the new Q5 enzyme is delivered, arrange for two teams to re-amplify the fragment that failed the amplification.
7.31 summary
1. UGT33 colony PCR, the result is positive
2. 4HPAAS gel recovery
3. Escherichia coli carrying UGT33
4. Take the plasmid and prepare for the subsequent pruning/transfer assay to detect whether it is a PUC-HA of ugt33 plasmid.
On August 3, the experiment concluded that UGT-33 PUC-HA carried out colony PCR and no positive bands.
2019.8.5. Summary
Realize the use of recombinant primers to amplify the RrUGT33 gene fragment and construct a PUC-4HPAAS-FLAG vector.
2019.8.6. Summary
1. Provincial implementation of plasmid preparation, collection
2. preparation of plasmids
2019.8.7. Plan.
Extract the 4HPAAS plasmid.
8.14
Make LB culture medium
8.15
1.Heat shock transformation of gold and bronze parts. (DH5 alpha of E. coli)
8.16
1. shake E. coli culture (3ml ×4 tubes) (gold and bronze parts)
8.17
1. plasmid extraction of gold and bronze parts. （failed）
2. Heat shock transformation of gold and bronze parts. (Transetta DE3 of E.coli)（canceled because we found out that Transetta DE3 bring Chl resistance.）.
8.18
1.make 2×YT sterile water and ddH2O.
2.shake E. coli culture (5ml LB culture with Chl ×2 tubes each) (gold and bronze parts)
8.19
1. plasmid re-extraction of gold and bronze parts.
2. Heat shock transformation of gold and bronze parts. (transBL 21 of E.coli) and make overnight culture in 37 degree Celsius.
3. plate cultivation (Chl and Kana resistance)
4. Confirm the presentation of Bronze Award Parts data, understand normalization, and communicate data processing with the modeling team
8.20
1. shake E. coli culture (3ml ×2 tubes) (bronze parts). (transBL 21 of E.coli)
2. make 5 different LB culture with PH value of 5,6,7,8, and 9.
3. 9L, 10C, due to the resistance error of No. 19 coating plate, use residual resuscitation bacterial solution coating plate in the morning
4. In the evening, 9L, 10C Bacterial Plate Bacteria, Select Monoclonal Shaker Bacteria
5. Send 1O, 8P, 9L, 10C, pSB1C3 (no sequencing results, inquire customer service, reply reason is low plasmid concentration)
6. Confirm the exact protocol of bronze medal.
8.21
1.In the morning, after 24 hours of frugal 1O culture, the concentration of bacterial solution reached a transferable shake.
2. Shake E. coli culture (100ml × 2tubes) (bronze parts) (transBL 21 of E.coli)
3. Save 1O sample continuously, measure OD600 to 0.6 to 0.8, then sample according to protocol time point to measure fluorescence (no effective fluorescence photo due to poor equipment condition)
8.23
Construct the vector of PUC-RrUGT33-HA successfully（use Gibson Assembly instead）.
8.24~ 9.22
1. Preparation and transfection of protoplasts from Arabidopsis thaliana.
2. Observe and measure protein expression rate by Western blot experiment.
3. Extraction and detection of target products (1.get cells expressing the target segments. 2. Conduct ethanol extraction. 3. detect target products by liquid chromatography)
9.22~
Compile and analyze result data.
