IGEM:Peking/2007/Count-Conjugation-Notebook/2007-8-12: Difference between revisions
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=oriT Knock Out by Liu Ting= | =oriT Knock Out by Liu Ting= | ||
==colony PCR test for DH5a-oriT-deleted R751 , DH5a-oriT-deleted F & DH5a-pUC18 with oriT-deleted pSC101 fragment== | ==colony PCR test for DH5a-oriT-deleted R751 , DH5a-oriT-deleted F & DH5a-pUC18 with oriT-deleted pSC101 fragment== | ||
===PCR system=== | ===PCR system(Every 200 ul is distributed to 10 tubes, that is, 20ul/PCR tube)=== | ||
*PCR system for DH5a-oriT-deleted R751 | *PCR system for DH5a-oriT-deleted R751 | ||
<pre> | <pre> | ||
pKO3-R(100uM) 2 ul | pKO3-R(100uM) 2 ul | ||
| Line 49: | Line 48: | ||
</pre> | </pre> | ||
*PCR system for DH5a-oriT-deleted F | *PCR system for DH5a-oriT-deleted F | ||
<pre> | <pre> | ||
F1S(20uM) 10 ul | F1S(20uM) 10 ul | ||
| Line 62: | Line 60: | ||
</pre> | </pre> | ||
*PCR system for DH5a-pUC18 with oriT-deleted pSC101 fragment | *PCR system for DH5a-pUC18 with oriT-deleted pSC101 fragment | ||
<pre> | <pre> | ||
S1S(100uM) 2 ul | S1S(100uM) 2 ul | ||
| Line 74: | Line 71: | ||
Total 200ul | Total 200ul | ||
</pre> | </pre> | ||
===PCR condition=== | ===PCR condition=== | ||
for all the above three: | for all the above three: | ||
Revision as of 22:32, 23 August 2007
Tandem Ori-T by Qu Mingzhi & Ren Ze
colony PCR Test for R751, pSC101(II)
- Test Lb- R751 plate, Lb- pSC101 have the correct plasmid.
- Test plate:LB- R751, Lb- pSB101, R751-pSC101 X Dh5α-R0040, R751 X Dh5α-R0040
- primer :R751 OriT primer, pSC101 primer.
- according to <Colony PCR STANDARD PROTOCOL>
- PCR system contains(each well):
0.5 µl Primer 1(100uM) 0.5 µl Primer 2 2 µl dNTP(2.5uM) 2.5 µl 10X Taq Buffer 0.25 µl Taq 19 µl dH20 1 µl template -------------------------- ~25 µl Total
- PCR program condition 1: 94℃ 5min, 94℃ 30s, 51℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end.
- PCR program condition 2: 94℃ 5min, 94℃ 30s, 55℃ 30s, 72℃ 45s, Go to step 2 for 29 times, 72℃ 10min, 4℃ end .
electrophorsis result
- top:from left to right
- 1-5 R751-pSC101 X Dh5α-R0040
- 6-10 R751 X Dh5α-R0040
- 11 R751
- 13 Maker(DL2000 plus)
- botton:from left to right
- 1-4 R751
- 5-10 psc101
- 11 Maker(DL2000 plus)
oriT Knock Out by Liu Ting
colony PCR test for DH5a-oriT-deleted R751 , DH5a-oriT-deleted F & DH5a-pUC18 with oriT-deleted pSC101 fragment
PCR system(Every 200 ul is distributed to 10 tubes, that is, 20ul/PCR tube)
- PCR system for DH5a-oriT-deleted R751
pKO3-R(100uM) 2 ul R1S(20uM) 10 ul dNTP(2.5uM) 4 ul 10X Taq Buffer 20 ul Taq 1 ul ddH2O 163ul (template) -------------------- Total 200ul
- PCR system for DH5a-oriT-deleted F
F1S(20uM) 10 ul F2A(20uM) 10 ul dNTP(2.5uM) 4 ul 10X Taq Buffer 20 ul Taq 1 ul ddH2O 155ul (template) -------------------- Total 200ul
- PCR system for DH5a-pUC18 with oriT-deleted pSC101 fragment
S1S(100uM) 2 ul S2A(100uM) 2 ul dNTP(2.5uM) 4 ul 10X Taq Buffer 20 ul Taq 1 ul ddH2O 171ul (template) -------------------- Total 200ul
PCR condition
for all the above three: 94℃ 10min; (94℃ 45s,51℃ 1min,72℃ 1min30s)/cycle X 30 cycles; 72℃ 10min; 16℃ 10min; end.
electrophorsis result
none...
