Skywalkerest (Talk | contribs) |
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<td style="text-align:center;vertical-align:middle;">Shuttle vector E. coli/B. meg; Protein production</td> | <td style="text-align:center;vertical-align:middle;">Shuttle vector E. coli/B. meg; Protein production</td> | ||
<td style="text-align:center;vertical-align:middle;">pSPAsp-hp was a gift from Dieter Jahn (Addgene plasmid # 48122)</td></tr> | <td style="text-align:center;vertical-align:middle;">pSPAsp-hp was a gift from Dieter Jahn (Addgene plasmid # 48122)</td></tr> | ||
− | <tr | + | <tr ><td style="text-align:center;vertical-align:middle;">pBE-2 </td> |
<td style="text-align:center;vertical-align:middle;">Shuttle vector E. coli/B. subtilis; Protein production</td> | <td style="text-align:center;vertical-align:middle;">Shuttle vector E. coli/B. subtilis; Protein production</td> | ||
<td style="text-align:center;vertical-align:middle;">pBE-2 was obtained from China Center for Type Culture Collection(CCTCC)</td></tr> | <td style="text-align:center;vertical-align:middle;">pBE-2 was obtained from China Center for Type Culture Collection(CCTCC)</td></tr> | ||
− | <tr | + | <tr ><td style="text-align:center;vertical-align:middle;">pBE-p43</td> |
<td style="text-align:center;vertical-align:middle;">Shuttle vector E. coli/B. subtilis; Protein production</td> | <td style="text-align:center;vertical-align:middle;">Shuttle vector E. coli/B. subtilis; Protein production</td> | ||
<td style="text-align:center;vertical-align:middle;">pBE-p43 was obtained from CCTCC</td></tr> | <td style="text-align:center;vertical-align:middle;">pBE-p43 was obtained from CCTCC</td></tr> | ||
− | <tr | + | <tr ><td style="text-align:center;vertical-align:middle;">pET28a </td> |
<td style="text-align:center;vertical-align:middle;">Protein production in E. coli</td> | <td style="text-align:center;vertical-align:middle;">Protein production in E. coli</td> | ||
<td style="text-align:center;vertical-align:middle;"> pET28a was obtained from Room 6133 of College of Life Sciences, Wuhan university(CLS-WHU)</td></tr> | <td style="text-align:center;vertical-align:middle;"> pET28a was obtained from Room 6133 of College of Life Sciences, Wuhan university(CLS-WHU)</td></tr> | ||
− | <tr | + | <tr ><td style="text-align:center;vertical-align:middle;">pET26b </td> |
<td style="text-align:center;vertical-align:middle;">Protein production in E. coli </td> | <td style="text-align:center;vertical-align:middle;">Protein production in E. coli </td> | ||
<td style="text-align:center;vertical-align:middle;">pET28a was obtained from Room 6135 of CLS-WHU</td></tr> | <td style="text-align:center;vertical-align:middle;">pET28a was obtained from Room 6135 of CLS-WHU</td></tr> | ||
Line 183: | Line 183: | ||
<h3 id="Primers">4.Primers</h3> | <h3 id="Primers">4.Primers</h3> | ||
<table border="1" align="center"> | <table border="1" align="center"> | ||
− | <tr bgcolor="#bdbbdb"><td style="vertical-align:middle;"></td> | + | <tr bgcolor="#bdbbdb"><td style="text-align:center;vertical-align:middle;">Primer names</td> |
− | <td style="vertical-align:middle;"></td></tr> | + | <td style="vertical-align:middle;">Sequence</td></tr> |
− | <tr><td style="vertical-align:middle;">Prefix-PartA-F</td> | + | <tr><td style="text-align:center;vertical-align:middle;">Prefix-PartA-F</td> |
<td style="vertical-align:middle;">5’- TCTAGAGGCCGCTGCGATCCCGCGAAG-3’</td></tr> | <td style="vertical-align:middle;">5’- TCTAGAGGCCGCTGCGATCCCGCGAAG-3’</td></tr> | ||
− | <tr><td style="vertical-align:middle;">PartA-RBS-R</td> | + | <tr><td style="text-align:center;vertical-align:middle;">PartA-RBS-R</td> |
<td style="vertical-align:middle;">5’- CTCCTCTTTAATCTCTAGACATCCTCTATGGTACTCGTGATGGCTTTATTG -3’</td></tr> | <td style="vertical-align:middle;">5’- CTCCTCTTTAATCTCTAGACATCCTCTATGGTACTCGTGATGGCTTTATTG -3’</td></tr> | ||
− | <tr><td style="vertical-align:middle;">RBS-Rdh-F</td> | + | <tr><td style="text-align:center;vertical-align:middle;">RBS-Rdh-F</td> |
<td style="vertical-align:middle;">5’- TCTAGAGATTAAAGAGGAGAAATACTAGATGAGACTTTACAGCAATAGAGATAGACCGAA-3’</td></tr> | <td style="vertical-align:middle;">5’- TCTAGAGATTAAAGAGGAGAAATACTAGATGAGACTTTACAGCAATAGAGATAGACCGAA-3’</td></tr> | ||
− | <tr><td style="vertical-align:middle;">Rdh-Ter-R</td> | + | <tr><td style="text-align:center;vertical-align:middle;">Rdh-Ter-R</td> |
<td style="vertical-align:middle;">5’- GTTTTATTTGATGCCTGGTTATTATCCCGCTGATGATTTAAGTCTTG-3’</td></tr> | <td style="vertical-align:middle;">5’- GTTTTATTTGATGCCTGGTTATTATCCCGCTGATGATTTAAGTCTTG-3’</td></tr> | ||
− | <tr><td style="vertical-align:middle;">Rdh-Ter-F</td> | + | <tr><td style="text-align:center;vertical-align:middle;">Rdh-Ter-F</td> |
<td style="vertical-align:middle;">5’- ATCATCAGCGGGATAATAACCAGGCATCAAATAAAACGAAAG-3’</td></tr> | <td style="vertical-align:middle;">5’- ATCATCAGCGGGATAATAACCAGGCATCAAATAAAACGAAAG-3’</td></tr> | ||
− | <tr><td style="vertical-align:middle;">Ter-Postfix-R</td> | + | <tr><td style="text-align:center;vertical-align:middle;">Ter-Postfix-R</td> |
<td style="vertical-align:middle;">5’- CTGCAGCGGCCGCTACTAGTATATAAACGCAGAAAGGC -3’</td></tr> | <td style="vertical-align:middle;">5’- CTGCAGCGGCCGCTACTAGTATATAAACGCAGAAAGGC -3’</td></tr> | ||
− | <tr><td style="vertical-align:middle;">pSPAsp-hp-BsrG1-F</td> | + | <tr><td style="text-align:center;vertical-align:middle;">pSPAsp-hp-BsrG1-F</td> |
<td style="vertical-align:middle;">5’- AAGGAGGTGAATGTACAATGAGACTTTACAGCAATAGAGATAGACCGAAT -3’</td></tr> | <td style="vertical-align:middle;">5’- AAGGAGGTGAATGTACAATGAGACTTTACAGCAATAGAGATAGACCGAAT -3’</td></tr> | ||
− | <tr><td style="vertical-align:middle;">pSPAsp-hp-BamH1-R</td> | + | <tr><td style="text-align:center;vertical-align:middle;">pSPAsp-hp-BamH1-R</td> |
<td style="vertical-align:middle;">5’- GCCGGTACCGGATCCTTATTATCCCGCTGATGATTTAA -3’</td></tr> | <td style="vertical-align:middle;">5’- GCCGGTACCGGATCCTTATTATCCCGCTGATGATTTAA -3’</td></tr> | ||
− | <tr><td style="vertical-align:middle;">pHIS1525-BsrG1-F</td> | + | <tr><td style="text-align:center;vertical-align:middle;">pHIS1525-BsrG1-F</td> |
<td style="vertical-align:middle;">5’- AAAGGGGGAAATGTACAATGAGACTTTACAGCAATAGAGATAGACCG-3’</td></tr> | <td style="vertical-align:middle;">5’- AAAGGGGGAAATGTACAATGAGACTTTACAGCAATAGAGATAGACCG-3’</td></tr> | ||
− | <tr><td style="vertical-align:middle;">pHIS1525-BamH1-R</td> | + | <tr><td style="text-align:center;vertical-align:middle;">pHIS1525-BamH1-R</td> |
<td style="vertical-align:middle;">5’- CCGGTACCGGATCCTTATTATCCCGCTGATGATTT-3’</td></tr> | <td style="vertical-align:middle;">5’- CCGGTACCGGATCCTTATTATCCCGCTGATGATTT-3’</td></tr> | ||
− | <tr><td style="vertical-align:middle;">pHIS1525-2-F</td> | + | <tr><td style="text-align:center;vertical-align:middle;">pHIS1525-2-F</td> |
<td style="vertical-align:middle;">5’- AAGGGGGAAATGTACAATGAGACTTTACAGCAATAGAGATAGACCG-3’</td></tr> | <td style="vertical-align:middle;">5’- AAGGGGGAAATGTACAATGAGACTTTACAGCAATAGAGATAGACCG-3’</td></tr> | ||
− | <tr><td style="vertical-align:middle;">pHIS1525-2-R</td> | + | <tr><td style="text-align:center;vertical-align:middle;">pHIS1525-2-R</td> |
<td style="vertical-align:middle;">5’- CCGGTACCGGATCCGTCCCGCTGATGATTT-3’</td></tr> | <td style="vertical-align:middle;">5’- CCGGTACCGGATCCGTCCCGCTGATGATTT-3’</td></tr> | ||
− | <tr><td style="vertical-align:middle;">pSPAsp1-F</td> | + | <tr><td style="text-align:center;vertical-align:middle;">pSPAsp1-F</td> |
<td style="vertical-align:middle;">5’- AAGGAGGTGAATGTACAATGAGACTTTACAGCAATAGAGATAGACCGAAT-3’</td></tr> | <td style="vertical-align:middle;">5’- AAGGAGGTGAATGTACAATGAGACTTTACAGCAATAGAGATAGACCGAAT-3’</td></tr> | ||
− | <tr><td style="vertical-align:middle;">pSPAsp1-R</td> | + | <tr><td style="text-align:center;vertical-align:middle;">pSPAsp1-R</td> |
<td style="vertical-align:middle;">5’- GCCGGTACCGGATCCGTCCCGCTGATGATTTAA-3’</td></tr> | <td style="vertical-align:middle;">5’- GCCGGTACCGGATCCGTCCCGCTGATGATTTAA-3’</td></tr> | ||
</table> | </table> |
Revision as of 01:25, 30 October 2017
Materials
1.Solutions
Solutions | Ingredients | Sterilization |
2x AB3(Antibiotic Medium No.3) | 35g/L AB3(DIFCO) in dH2O | Autoclave at 121℃ for 20 min |
2×SMM | 40mM Maleic acid, 80mM NaOH, 40mM MgCl2•6H2O, 1M Sucrose in dH2O | Filtration |
PEG-P | 400g/L PEG-6000 in 1x SMM | Autoclave at 121℃ for 11 min |
SMMP | 2x AB3 : 2×SMM= 1:1 | Separately sterilized |
Solution A | 206g/L Sucrose, 13g/L MOPS, 1.2g/L NaOH in dH2O.Adjust to pH 7.3 with NaOH | Filtration |
Solution B | 14.04g/L Agar, 0.70g/L Casamino acids, 35.09g/L Yeast Extract in dH2O | Autoclave at 121℃ for 15 min |
8x CR5 salts | 2g/L K2SO4, 80g/L MgCl2•6H2O, 0.4g/L KH2PO4, 17.6g/L CaCl2 in dH2O | Autoclave at 121℃ for 15 min |
12% Proline 120g/L | L-proline in dH2O | Filtration |
20% Glucose | 200g/L Glucose in dH2O | Filtration |
CR5-top agar(2.5ml) | 1.25ml Solution A, 288μl CR5 salts, 125μl 12% Proline, 125μl 20% Glucose, 713μl Solution B(42℃) | Separately sterilized and mix in order |
Lysis buffer | 100mM Na3PO4, 5mg/ml Lysozyme. Adjust to pH 6.3 with H3PO4. 1μl 1 M MgSO4 solution and 2 μl HS-Nuclease* (250 U/μl, cat.# GENUC10700-01, final concentration of 500 U/ml) | Filtration |
(Other basic molecular biological materials are not listed here)
2.Plasmids
Plasmids | Purposes | Cite/Source |
pSPAsp-hp | Shuttle vector E. coli/B. meg; Protein production | pSPAsp-hp was a gift from Dieter Jahn (Addgene plasmid # 48122) |
pBE-2 | Shuttle vector E. coli/B. subtilis; Protein production | pBE-2 was obtained from China Center for Type Culture Collection(CCTCC) |
pBE-p43 | Shuttle vector E. coli/B. subtilis; Protein production | pBE-p43 was obtained from CCTCC |
pET28a | Protein production in E. coli | pET28a was obtained from Room 6133 of College of Life Sciences, Wuhan university(CLS-WHU) |
pET26b | Protein production in E. coli | pET28a was obtained from Room 6135 of CLS-WHU |
3.Bacteria
Strains | Purposes | Cite/Source |
Escherichia coli DH5α | Plasmid replication, Protein production | E. coli DH5α was obtained from Room 6133 of CLS-WHU |
Bacillus subtilis 168 | Protein production | B. subtilis 168 was obtained from CCTCC |
Bacillus megaterium DSM B32 | Protein production | B. megaterium DSM B32 was obtained from CCTCC |
Nitratireductor pacificus pht-3B | Genome PCR | Nitratireductor pacificus pht-3B was obtained from CCTCC |
4.Primers
Primer names | Sequence |
Prefix-PartA-F | 5’- TCTAGAGGCCGCTGCGATCCCGCGAAG-3’ |
PartA-RBS-R | 5’- CTCCTCTTTAATCTCTAGACATCCTCTATGGTACTCGTGATGGCTTTATTG -3’ |
RBS-Rdh-F | 5’- TCTAGAGATTAAAGAGGAGAAATACTAGATGAGACTTTACAGCAATAGAGATAGACCGAA-3’ |
Rdh-Ter-R | 5’- GTTTTATTTGATGCCTGGTTATTATCCCGCTGATGATTTAAGTCTTG-3’ |
Rdh-Ter-F | 5’- ATCATCAGCGGGATAATAACCAGGCATCAAATAAAACGAAAG-3’ |
Ter-Postfix-R | 5’- CTGCAGCGGCCGCTACTAGTATATAAACGCAGAAAGGC -3’ |
pSPAsp-hp-BsrG1-F | 5’- AAGGAGGTGAATGTACAATGAGACTTTACAGCAATAGAGATAGACCGAAT -3’ |
pSPAsp-hp-BamH1-R | 5’- GCCGGTACCGGATCCTTATTATCCCGCTGATGATTTAA -3’ |
pHIS1525-BsrG1-F | 5’- AAAGGGGGAAATGTACAATGAGACTTTACAGCAATAGAGATAGACCG-3’ |
pHIS1525-BamH1-R | 5’- CCGGTACCGGATCCTTATTATCCCGCTGATGATTT-3’ |
pHIS1525-2-F | 5’- AAGGGGGAAATGTACAATGAGACTTTACAGCAATAGAGATAGACCG-3’ |
pHIS1525-2-R | 5’- CCGGTACCGGATCCGTCCCGCTGATGATTT-3’ |
pSPAsp1-F | 5’- AAGGAGGTGAATGTACAATGAGACTTTACAGCAATAGAGATAGACCGAAT-3’ |
pSPAsp1-R | 5’- GCCGGTACCGGATCCGTCCCGCTGATGATTTAA-3’ |
(Not all the primers are listed here but some important ones)