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Team:Imperial College/Transformation Results
From 2008.igem.org
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*The protocol was carried out with two repeats and it was noticed that similar numbers of transformants were yielded for both repeats.}} | *The protocol was carried out with two repeats and it was noticed that similar numbers of transformants were yielded for both repeats.}} | ||
| - | + | {{Imperial/Box1|Transformation Protocol 2| | |
'''[http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Wetlab/Transformation_protocol_4 Click this link for Transformation protocol 2 '''] | '''[http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Wetlab/Transformation_protocol_4 Click this link for Transformation protocol 2 '''] | ||
<br> | <br> | ||
| - | + | '''Brief Descrption of protocol'''<br> | |
Preparation of Competent Cells: | Preparation of Competent Cells: | ||
*Start an overnight culture, | *Start an overnight culture, | ||
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*Defrost cells and electroporate with DNA, | *Defrost cells and electroporate with DNA, | ||
*Plate the cells onto a LB agar plate with suitable antibiotics. | *Plate the cells onto a LB agar plate with suitable antibiotics. | ||
| - | + | <br> | |
| - | + | '''Results'''<br> | |
*We performed a transformation using the integration plasmid pDR110. Two repeats were carried out using this protocol. For the two repeats the competent cells were grown to an O.D.<sub>600</sub> of 1.5 and 1.8 and transformed with a range of DNA concentrations from 0 to 400ng of DNA. Transformants were selected with the antibiotic streptinomycin, the resistance of which is encoded by the pDR110. | *We performed a transformation using the integration plasmid pDR110. Two repeats were carried out using this protocol. For the two repeats the competent cells were grown to an O.D.<sub>600</sub> of 1.5 and 1.8 and transformed with a range of DNA concentrations from 0 to 400ng of DNA. Transformants were selected with the antibiotic streptinomycin, the resistance of which is encoded by the pDR110. | ||
*The results from this protocol were mixed, for the first repeat there were transformed colonies on the plate of ''B.subtilis'' transformed with no DNA. The most likely cause for this is that there was a contamination in preparation of these samples. For the second repeat no transformants was seen for the ''B.subtilis'' transformed with no DNA, showing that there was no contamination. | *The results from this protocol were mixed, for the first repeat there were transformed colonies on the plate of ''B.subtilis'' transformed with no DNA. The most likely cause for this is that there was a contamination in preparation of these samples. For the second repeat no transformants was seen for the ''B.subtilis'' transformed with no DNA, showing that there was no contamination. | ||
| - | *Comparison of the different efficiencies of transformation between experiments showed that the number of transformants was highly variable. The cause for this is thought to be the settings for electroporation equipment. We able to control the voltage and the resistance but not the pulse length. Previous studies have shown that a pulse length below 5 msec gives low efficiency of transformation. A maximum efficiency is found for a pulse legnth of 10 msec. Between our two repeats we found a range of pulse lengths between 3-11 msec and as a result variability in the number of transformed ''B.subtilis''. | + | *Comparison of the different efficiencies of transformation between experiments showed that the number of transformants was highly variable. The cause for this is thought to be the settings for electroporation equipment. We able to control the voltage and the resistance but not the pulse length. Previous studies have shown that a pulse length below 5 msec gives low efficiency of transformation. A maximum efficiency is found for a pulse legnth of 10 msec. Between our two repeats we found a range of pulse lengths between 3-11 msec and as a result variability in the number of transformed ''B.subtilis''.}} |
Revision as of 23:55, 27 October 2008
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