Team:XJTLU-CHINA/Notebook
Frequently used Protocols
Electrophoresis DNA Gel Extraction
Work Flow Gene Synthesis Gibson Assembly
PCR Amplification
of DNA Fragment from
Plasmid Transformation SDS-PAGE RT-PCR
Fluorescence
measurement Colony PCR RNA extraction Gel extraction
March:
Overview:
Fragments construction.
3.14
Group 1:
Test of PCR standard for tsf & tufA gene
3.15
Group 1:
Gel electrophoresis of tsf gene and tufA gene
Gel extraction of tsf
TfuA gene PCR (test the optimal condition
Gel electrophoresis of tufA gene
3.17
Group 1:
Plasmid extraction
Isolation of plasmid pACYCDuet from Top.10 cells
3.23
Group 1:
Extraction of QBeta genome
3.26
Group 1:
Transformation of pQBeta7 to competent cell Top.10
3.27
Group 1:
Incubate transformed competent cells
April:
Overview:
Fragments construction.
Ligation of genome fragments.
4.19
Group 1: Tu PCR.
4.20
Group 1: Tu PCR.
Group 3: gel extraction.
4.25
Group 1: do the pcr purification for Tu gene
4.26
Group 1: Gel extraction.
Group 3: ligate genome fragment 1 and 2.
4.27
Group 3: Gel extraction.
4.28
Group 1: Rep PCR.
June:
Overview:
Recombination of plasmids.
6.23
Group 4:
Transformation for QBeta 7 Select HB107 with PUC19 plasmids and cultivation Result: no rep has been detected. Construction of pACYC-TS
1. PCR of tsf Result: tsf has been coamplified 2. Electrophoresis to examine the length of tsf Result: tsf has correct (expected length) 3. PCR purification 4. pACYC and tsf double digestion with NcoI and BamHI 5. Ligation of pACYC(digested and tsf (digested Result: the mixture might contain pACYC-ts 6. Transformation of pACYC-ts
6.25
Goup 1:
Check the fragment inserted in pACYC 1. Cell amplification 2. PCR of a fragment on pACYC containing ts 3. Gel electrophoresis 4. Second turn PCR
6.26
Group 1:
Tuf PCR 1. Tu nesting PCR 2. Gel electrophoresis of the PCR product 3. Tu nesting PCR product purification 4. Tu PCR 5. Tel electrophoresis of the PCR product
6.27
Group 4:
HB101 selection and cultivation 1. Select HB101 with PUC19 plasmids and cultivation 2. PCR for testing the product of HB101 3. Select A1¬，B2, C3, D1, E3, F2¬ tubes which contain HB101 with pUC19 into conical flask 4. Cultivate over night
6.28
Group 1:
Ligation of Tu and Transformation of pACYC-tu 1. Double digestion of tu and pACYC 2. Ligation of tu onto pACYC 3. Transformation
6.29
Group 1:
Check whether tu has been inserted into pACYC 1. Cell amplification 2. Tu testing PCR 3. Gel electrophoresis
6.30
Group 1:
Ligation of tu & transformation of pACYC-tu 1. Ligation of tu onto pACYC 2. Transformation
July:
Overview:
Fragments PCR.
Recombination of plasmids.
Ligation of genome fragments.
7.2
Group 1:
Digestion of plasmid pACYC-DuetI and DNA tufa & ligation and transformation of pACYC-tu
1. double digestion of tu & pACYC
2. Clean up of digested pACYC-DuteI and tufa
3. Ligation of pACYC-DuetI with tfuA
4. Transformation of pACYC-tu into Top-10 cells
7.4
Group 4:
Transformation fot Duet (Duet to BL21
Check whether tfuA is inserted into pACYC-DuetI
1. Cell amplification
2. Tu testing PCR
3. Gel electrophoresis Results: none of the colonies selected contain s the recombinant plasmid pACYC-tu
7.5
Group 1:
Amplification of E.coli cells containing pACYC-ts
Group 4:
Double transformation for duet and acyc
7.6
Group 1:
Digest pETDuet-1 with enzymes and test
Gibson assembly of intron a, b and kan
Run electrophoresis to test
Clean up
7.7
Group 1:
Digestion of plasmid pACYC-DyetI and DNA tufa & ligation and transformation of pACYC-tu
1. Digestion and ligation of pACYC-DuetI and tufa (same as the recording on 7.2
Transformation of recombinant plasmids into Top.10 cells
Group 4:
Part II
1. Digest Duet with enzyme
2. Clean up
3. Gibson assembly
4.Transformation to BL21
7.8
Group 1:
PCR testing of tu insert
1. Cell amplification
2. Tu testing PCR (same as the recording on 7.4
3. Gel electrophoresis
Results: A6 has the target length of band
4. Amplification
Result: 600nm (Abs: 0.103
Group 4:
Part II
1. Gibson assembly: intron+ kana
2. Run gel electrophoresis to test
Result: intron a_ kan was not connected
Intron b+ kan connected a little
3. PCR the bacteria BL21 contain recombinant Duet-1 (Target+ Ndel & Xhot
7.9
Group 1:
Mini prep and planting of pACYC-tu (check pACYC-tu is the only plasmid
1. Planting of transformed Top.10 cells
2. Miniprep of plasmids
Re-testing by PCR of plasmid pACYC-tu (check whether pACYC-tu is the only plasmid
1. PCR testing of ty insert
2. Gel electrophoresis
Results: there is the target insert
Transformation of pACYC-tu into Top.10 E.coli cells
7.10
Group 1:
Amplification of Top.10 E.coli cells containing pACYC-tu
7.11
Group 4:
PCR testing of pACYC
Gel electrophoresis
Digest Duet-1 with Xbal &AfI III
Clean up
Gibson assembly
Transformation Gibson assembly product into BL21
7.12
Group 4:
PCR the bacteria BI21 contain pACYC, intron a-kan-intron b-DuetL(group 1
7.14
Group 4:
Digest Duet-1 with Xbal and Afl II
Clean up
Gel electrophoresis
Gibson assembly
Transformation
7.15
Group 4:
PCR the bacteria Top.10 contain intron a-kan-intron b+-Duet-1(Xbal & afl II
7.25
Group 1:
Construction of pACYC-tu-ts
1. Double digestion with NcoK and BamHI
2. Ligation of tsf onto pACYC-tu
3. PCR for tsf amplification from pACYC-ts
Result: tsf has been amplified successfully
Group 2:
Use bigson assembly ligation genome 1, 2, 3, 4
PCR to replicase genome 1, 2, 3, 4 (S15 system
Group 3:
Digest Duetl WITH Xbal and afl III
Gel electrophoresis
Clean up
Gibson assembly
Clean up
PCR reaction of a+b+k
Gel electrophoresis
Clean up a+b+k
Assemble a+b+k+Duet-1 with Gibson assembly kid
Clean up a+b+k+Duet-1
Transform plasmids (step 9 into Top.10, then plate them onto AMP plates
7.26
Group 1:
Construction of pACYC-tu-ts
1. Double digestion of the pACYC-tu with NcoL and BamHI
2. Ligation of tsf onto pACYC-tu
Group 2:
Amplify the Gibson product (1-2-3-4
PCR
Gel electrophoresis
Bacterial glycerol stock
Extract plasmids
Group 4:
Gibson assembly:
Clean up:
PCR a+k
Pick up colonies from two AMP plates made on 7.24
Inculbate 35 colonies under 37℃ over 2 hours
PCR
Gel electrophoresis
7.27
Group 1:
Ligation & transformation
1. Continue the process of ligation after over night incubation at 16℃
2. Transformation of pACYC-tu-ts
Group 2:
PCR genome 1~4 from plasmid, which was entracted from Top.10
Gel electrophoresis
Results: sample1 and 4 has similar gel electrophoresis result
Group 4:
Get the PCR product +(a+k
Agarose gel electrophoresing of maturase
Gel electrophoresis
Gel electrophoresis
Clean up
7.28
Group 1:
Check the inserted fragment of tsf in pACYC-tu-ts
1. Cell amplification
2. PCR to check the fragment on pACYC-tu-ts to see whether ts can be generated with taq enzyme. 30 tubes were used
3. Gel electrophoresis to see the results from PCR tsf amplification
Result: no target length of the fragments appeared. Experiment need to be repeated.
7.29
Group 1:
Check the inserted fragment of tsf in pACYC-tu-ts (the same procedure as in the experiment on 7.28
Result: still no target length of the fragments appeared
Group 4:
PCR testing of pACYC
Gel electrophoresis
Clean up and nanodrop
PCR the plasmids from bacteria Top.10 contain a-kan-b-Deut-1: D2, B6
PCR testing of pACYC
August:
Overview:
Recombination of plasmids.
Ligation of genome fragments.
8.1
Group 1:
Check the insert of tsf in pACYC-tu (same as 7.28) Excluding gel electrophoresis
Group 4:
Gibson assembly
PCR the reaction of a+k (Q5
Gel electrophoresis+ cleanup
PCR the reaction of a+k (S15
8.2
Group 4:
Extract the gel containing a+k (S15)
Gel electrophoresis
Gel extraction
Check the pACYC+ matuase in bacteria
Cell amplification
PCR reaction for each colonies
Gel electrophoresis
PCR: knock our for plasmid in acyc
8.3
Group 4:
Gel electrophoresis of the knock our PCR product
PCR reaction for intron a and kan
Gel electrophoresis
Gibson assembly of intron a and kan
Group 1:
Plasmids (pACYC-ts-tu)
Result: concentration of nucleic acid: 168.1ng/Μl 260/280: 1.92
8.4
Group 1:
Gel electrophoresis of rep fragment from PCR
PCR purification: use QIA quick PCR purification kid (250
Double digestion of Rep and PET-Duet-1
Incubate at 37℃ waterbath for 2 hours
Use MiniElute reaction ceanup kid (QIAGEN Cat No.28204
Ligation of pETDuet-I and Rep
Incubate at 16℃ incubator for overnight
Group 4:
PCR reaction for intron b. (412
Gel electrophoresis
Clean up
Check mutuase in bacteria
Cell amplification
PCR for each colonies
Gel electrophoresis
PCR reaction for intron b
8.5
Group 4:
Gel electrophoresis
PCR of matuase
Transformation of ligation mixture (over night at 16 degrees）
8.8
Group 4:
Geo eoectrophoresis
Knock out T7 promotor of pACYC
Gel electrophoresis
Clean up
8.9
Group 1:
construction of pACYC-tu-ts and transformation of the recombinant plasmid into the competent cells
1. Double digestion of pACYC-tu and tsf
2. Ligation of tsf onto pACYC-tu
3. Transformation
Group 4:
Transformation of pACYC-T7 and maturase into Top.10
Gel electrophoresis
Clean up
8.10
Group 1:
Check whether ts has been inserted in pACYC-tu
Cell amplification
PCR with the amplified cells as template
Construction of pETDuet-Rep
Transformation of pETDuet into Top.10
Group 4:
Gel electrophoresis of PCR products: K1, K2, A1, A2
Clean up
Gel electrophoresis
Digest Duet-1-target with Xbal and Afl III
PCR: 8.10 PCR of intron A
8.11 PCR of kan sample
PCR of intron b
Knock out T7 promotor of pACYC
Gel electrophoresis clean up and nanodrop
8.11
Group 4:
Gibson assembly
Transformation of M-pACYC-T7 into Top.10
Digest Duet-1-target with Afl III and Xbal
Gibson assembly
PCR of a+b+k
8.12
Group 1:
pETDuet-Rep testing
group 4:
Gel electrophoresis of PCR product
Clean up
Digest Duet-target with Afl III and Xbal
Gel electrophoresis
Clean up
PCR of a+b+k
PCR OF Duet + target
Gel electrophoresis
8.13
Group 1:
Redo the experiment in 8.12 procedures
Result: no expected bands presented in electrophoresis gel
pET Duet-Rep testing using S15
8.17
Group 4:
PCR of intron a.
Gel electrophoresis
Clean up
Gibson assembly
Gel electrophoresis
PCR of Gibson products
PCR of intron a
8.18
Group 4:
Gibson assembly
Mix
Gel electrophoresis
8.19
Group 4:
Gel extraction: k+b: 23.5ng/μL
8.20
Group 4:
Digest Duet-1 with Xbal and Afl II
Clean up
Gibson assembly
Transformation of Gibson
Gibson: gel extraction
8.21
Group 4:
PCR the bacteria Top.10 contain Duet+ akb
Gel electrophoresis
Agarose gel electrophoresis
After clean up
The concentraction: 202.2ng/μL
260/280： 1.84
8.22
Group 4:
Gibson assembly
Transformation to Top.10
Gibson assbmely
Clean up
OCR of k+b
Gel electrophoresis and gel extraction
Digest pETDuet-1 with Xbal and Afl III
Gel electrophoresis
Clean up
Digest Duet-target with Xbal and Afl III
Gel electrophoresis
Result: no target sequence
8.23
Group 4:
PCR the bacteria Top.10 contain pACYC-ts and maturase
Gel electrophoresis
Digestion of maturase-ACYC plasmid
8.24
Group 4:
PCR of kan sample
PCR of intron b
Overlap PCR of kan+intron b
Gel electrophoresis
Clean up
8.25
Group 4:
PCR of intron a (plasmid to get intron a (enlonged
Gel electrophoresis
Result: no target sequence
PCR the bacteria Top.10 contain pACYC+maturase
Gel electrophoresis
Gibson assembly
October:
Overview：
Verify the retrohoming function of Group 2 intron.
Measurement of Red florescence protein to compare the function of temperature controlled switch after mutation.
10.3
Group 4:
Obtain the pACYC plasmid containing LtrA protein.
Transformation of pACYC and PETduet into BL 21, then spread it into double-antibody plates with resistance of Amp and Cmr.
10.4
Group 4:
Select colonies cultured last night for incubation within LB broth.
Measure OD600 of bacteria fluid till it reaches 0.8 and Induce it with IPTG and arabinose.
Spread the induced bacteria fluid into both single-antibody plats with Kan and triple-antibody plates with resistance of Amp, Cmr and Kan.
10.5
Group 4:
Conduct the PCR and gel electrophoresis to check the retrohoming function of intron in single-antibody plats with Kan and triple-antibody plates with resistance of Amp,
Cmr and Kan.
Transformation of pACYC and PETduet into BL 21 and spread it into double-antibody plates with resistance of Amp and Cmr.
Single endonuclease digestion of pACYC plasmid.
RFP group:
Measure the Red florescence protein and OD600 of BL21 incubated for 18 hours at 26℃ and 37℃ respectively from the first pool.
10.6
Group 4:
Select colonies cultured last night for incubation within LB broth.
Measure OD600 of bacteria fluid till it reaches 0.8 and Induce it with IPTG and arabinose.
Spread the induced bacteria fluid into both single-antibody plats with Kan and triple-antibody plates with resistance of Amp, Cmr and Kan.
Conduct the PCR and gel electrophoresis to check the retrohoming function of intron in single-antibody plats with Kan and triple-antibody plates with resistance of Amp,
Cmr and Kan.
10.7
Group 4:
Reselectc colonies from single-antibody plats and the triple-antibody plates last night.
Conduct the PCR and gel electrophoresis to check the retrohoming function of intron.
Extract the plasmids from both single-antibody plats and the triple-antibody plates.
Double endonuclease digestion of the extracted plasmids.
RFP group:
Measure the Red florescence protein and OD600 of BL21 incubated for 18 hours at 26℃ and 37℃ respectively from the second pool.
10.8
Group 4:
Reselectc colonies from the plates in October.5 and induce.
Spread the induced bacteria fluid into both single-antibody plats with Kan and triple-antibody plates with resistance of Amp, Cmr and Kan.
Conduct the PCR and gel electrophoresis to check the retrohoming function of intron in single-antibody plats with Kan and triple-antibody plates with resistance of Amp,
Cmr and Kan.
10.9
Group 4:
Extract the plasmids from both single-antibody plats and the triple-antibody plates.
Double endonuclease digestion of the extracted plasmids.
RFP group:
Select 10 colonies from the second pool.
Measure the Red florescence protein and OD600 of BL21 incubated for 18 hours at 26℃ and 37℃ respectively.
10.10
Group 4:
Transformation of pACYC and PETduet into BL 21, then spread it into double-antibody plates with resistance of Amp and Cmr.
Spread the induced bacteria fluid into both single-antibody plats with Kan and triple-antibody plates with resistance of Amp, Cmr and Kan.
10.11
Group 4:
Conduct the PCR and gel electrophoresis to check the retrohoming function of intron in single-antibody plats with Kan and triple-antibody plates with resistance of Amp,
Cmr and Kan.
Extract the plasmids from both single-antibody plats and the triple-antibody plates.
Conduct the PCR and gel electrophoresis to check the retrohoming function of intron using the extracted plasmids.
10.12
Group 4:
Double endonuclease digestion of the extracted plasmids.
Reselect colonies to conduct the PCR and gel electrophoresis, checking the retrohoming function of intron in single-antibody plats with Kan and triple-antibody plates with
resistance of Amp, Cmr and Kan.
10.13
Group 4:
Transformation of pACYC and PETduet into BL 21, then spread it into double-antibody plates with resistance of Amp and Cmr.
Spread the induced bacteria fluid into both single-antibody plats with Kan and triple-antibody plates with resistance of Amp, Cmr and Kan.
Conduct the PCR and gel electrophoresis to check the retrohoming function of intron in single-antibody plats with Kan and triple-antibody plates with resistance of Amp,
Cmr and Kan.
10.14
Group 4:
Extract the plasmids from both single-antibody plats and the triple-antibody plates.
Double endonuclease digestion of the extracted plasmids.
10.15
Group 4:
Transformation of pACYC and PETduet into BL 21, then spread it into double-antibody plates with resistance of Amp and Cmr.
Spread the induced bacteria fluid into both single-antibody plats with Kan and triple-antibody plates with resistance of Amp, Cmr and Kan.
10.16
Group 4:
Extract the plasmids from both single-antibody plats and the triple-antibody plates.
Conduct the PCR and gel electrophoresis to check the retrohoming function of intron using the extracted plasmids.
Double endonuclease digestion of the extracted plasmids.
