IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-16: Difference between revisions
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*We tried all kinds of conditions and the damn PCR failed again and again! I guessed it was because of the unbalanced PCR primers. However scince I need a 30 bp overlap to link the two PCR fragments together (via overlapped PCR), I didn't have another choice in primers' sequences. | *We tried all kinds of conditions and the damn PCR failed again and again! I guessed it was because of the unbalanced PCR primers. However scince I need a 30 bp overlap to link the two PCR fragments together (via overlapped PCR), I didn't have another choice in primers' sequences. | ||
#I was tired of colony PCR and designed to do chromasome extraction and use them as template, to avoid as much protein (in bacteria) as possible. | #I was tired of colony PCR and designed to do chromasome extraction and use them as template, to avoid as much protein (in bacteria) as possible. | ||
==Chromasome Extraction== | ==Chromasome Extraction== | ||
#Picked a single colony from plates and shake overnight in 5 mL LB, 37 centigrade. | #Picked a single colony from plates and shake overnight in 5 mL LB, 37 centigrade. | ||
#As control, culture another sample in 30 centigrade (An unreasonable requirement from CGMCC). | #As control, culture another sample in 30 centigrade (An unreasonable requirement from CGMCC). | ||
*Cells can grow up in LB medium with 37 centigrade, overnight. Still cannot make sure whether the conjugation plasmids are in the grown cells because of the PCR failure. | |||
== | ==To-do List== | ||
Use chromosome extraction template, Taq and Pfu enzyme to try again. Plan to re-design primers in case it fails again. | |||
=Plasmid Construction= | |||
==Eletrophoresis of Extracted Plasmid== | ==Eletrophoresis of Extracted Plasmid== | ||
*By Ma and Yu Tao. | *By Ma and Yu Tao. | ||
Revision as of 22:28, 14 August 2007
oriT Knock Out
- By Xu Anting and Liu Ting
What will you do after endless failure...?
- Cannot repeat the PCR result of oriT in F/R751/pSC101. In the four trials with various conditions, F bands appeared twice, R751 once, and pSC101 twice.
- We tried all kinds of conditions and the damn PCR failed again and again! I guessed it was because of the unbalanced PCR primers. However scince I need a 30 bp overlap to link the two PCR fragments together (via overlapped PCR), I didn't have another choice in primers' sequences.
- I was tired of colony PCR and designed to do chromasome extraction and use them as template, to avoid as much protein (in bacteria) as possible.
Chromasome Extraction
- Picked a single colony from plates and shake overnight in 5 mL LB, 37 centigrade.
- As control, culture another sample in 30 centigrade (An unreasonable requirement from CGMCC).
- Cells can grow up in LB medium with 37 centigrade, overnight. Still cannot make sure whether the conjugation plasmids are in the grown cells because of the PCR failure.
To-do List
Use chromosome extraction template, Taq and Pfu enzyme to try again. Plan to re-design primers in case it fails again.
Plasmid Construction
Eletrophoresis of Extracted Plasmid
- By Ma and Yu Tao.
- J23066, pSB3K3, pSB1AK3, R0010, J61003 are all tested. (Without Digestion.)
- Each of R0010, J61003 and pSB1AK3 has a single band. (Weird) And pSB3K3 and J23066 have no bands.
- Notes: This eletrophoresis is especially for me to better understand the essence of electrophoresis, to make better use of the DNA dye and to adjust the apparatus to a better condition. Besides, there is still something wrong with our UVP Bioimaging System. Therefore, the result of the electrophoresis cannot be shown.
Miniprep of E0040
- BY Yu Tao.
Eletrophoresis of Extracted Plasmid
- By Yu Tao.
- R0040, J01060, E0040 adn B0015 are tested. (Without Digestion.)
- All have a single band.
- Note:
Instruction of the DNA Dye
For conveniences, the DNA dye can be diluted 100 times by TAE first and stored at 4 centigrade degree. The final cencentration in the Sample can be 1000 times diluted.
Preculture E0040 J23066 and pSB3K3 for Miniprep Tomorrow=
- By Yu Tao.
