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TUDelft/24 October 2008
From 2008.igem.org
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Today we sent our lyophilized DNA to MIT. We also sent a number of strains to Baseclear (NL) for sequencing. | Today we sent our lyophilized DNA to MIT. We also sent a number of strains to Baseclear (NL) for sequencing. | ||
| - | ==Protein | + | ==Protein Precipitation== |
Including yesterday's PCA precipitation, four different protein precipitation protocols were conducted to obtain protein content measurements without the disturbance of lysis buffer. The four protocols can be found [[Team:TUDelft/Protocols#Protein_Precipitation|here]], they are PerChloric Acid (PCA), TriChloroAcetic acid (TCA)/acetone, TCA/Deoxycholate (DOC) and methanol/chloroform precipitation. For each of these precipitation methods four samples of constructs BBa_K115012, BBa_K115035 and BBa_K115036 and the calibration curve (bovine serum albumin in H<sub>2</sub>O) were resuspended in 1x lysis buffer. After precipitation protocol and BCA assay was performed, standard calibration curves were made for all four precipitation protocols together with an untreated calibration curve for comparative reasons. The result can be seen in figure 1.<br/><br/> | Including yesterday's PCA precipitation, four different protein precipitation protocols were conducted to obtain protein content measurements without the disturbance of lysis buffer. The four protocols can be found [[Team:TUDelft/Protocols#Protein_Precipitation|here]], they are PerChloric Acid (PCA), TriChloroAcetic acid (TCA)/acetone, TCA/Deoxycholate (DOC) and methanol/chloroform precipitation. For each of these precipitation methods four samples of constructs BBa_K115012, BBa_K115035 and BBa_K115036 and the calibration curve (bovine serum albumin in H<sub>2</sub>O) were resuspended in 1x lysis buffer. After precipitation protocol and BCA assay was performed, standard calibration curves were made for all four precipitation protocols together with an untreated calibration curve for comparative reasons. The result can be seen in figure 1.<br/><br/> | ||
Latest revision as of 21:04, 29 October 2008
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| September | ||||||
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| [http://2008.igem.org/TUDelft/1_September_2008 1] | [http://2008.igem.org/TUDelft/2_September_2008 2] | [http://2008.igem.org/TUDelft/3_September_2008 3] | [http://2008.igem.org/TUDelft/4_September_2008 4] | [http://2008.igem.org/TUDelft/5_September_2008 5] | [http://2008.igem.org/wiki/index.php?title=TUDelft/6_September_2008&action=edit 6] | [http://2008.igem.org/wiki/index.php?title=TUDelft/7_September_2008&action=edit 7] |
| [http://2008.igem.org/TUDelft/8_September_2008 8] | [http://2008.igem.org/TUDelft/9_September_2008 9] | [http://2008.igem.org/TUDelft/10_September_2008 10] | [http://2008.igem.org/TUDelft/11_September_2008 11] | [http://2008.igem.org/TUDelft/12_September_2008 12] | [http://2008.igem.org/wiki/index.php?title=TUDelft/13_September_2008&action=edit 13] | [http://2008.igem.org/wiki/index.php?title=TUDelft/14_September_2008&action=edit 14] |
| [http://2008.igem.org/TUDelft/15_September_2008 15] | [http://2008.igem.org/TUDelft/16_September_2008 16] | [http://2008.igem.org/TUDelft/17_September_2008 17] | [http://2008.igem.org/TUDelft/18_September_2008 18] | [http://2008.igem.org/TUDelft/19_September_2008 19] | [http://2008.igem.org/wiki/index.php?title=TUDelft/20_September_2008&action=edit 20] | [http://2008.igem.org/wiki/index.php?title=TUDelft/21_September_2008&action=edit 21] |
| [http://2008.igem.org/TUDelft/22_September_2008 22] | [http://2008.igem.org/TUDelft/23_September_2008 23] | [http://2008.igem.org/TUDelft/24_September_2008 24] | [http://2008.igem.org/TUDelft/25_September_2008 25] | [http://2008.igem.org/wiki/index.php?title=TUDelft/26_September_2008&action=edit 26] | [http://2008.igem.org/wiki/index.php?title=TUDelft/27_September_2008&action=edit 27] | [http://2008.igem.org/wiki/index.php?title=TUDelft/28_September_2008&action=edit 28] |
| [http://2008.igem.org/TUDelft/29_September_2008 29] | [http://2008.igem.org/TUDelft/30_September_2008 30] | |||||
| October | ||||||
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| [http://2008.igem.org/TUDelft/1_October_2008 1] | [http://2008.igem.org/TUDelft/2_October_2008 2] | [http://2008.igem.org/TUDelft/3_October_2008 3] | [http://2008.igem.org/wiki/index.php?title=TUDelft/4_October_2008&action=edit 4] | [http://2008.igem.org/wiki/index.php?title=TUDelft/5_October_2008&action=edit 5] | ||
| [http://2008.igem.org/TUDelft/6_October_2008 6] | [http://2008.igem.org/TUDelft/7_October_2008 7] | [http://2008.igem.org/TUDelft/8_October_2008 8] | [http://2008.igem.org/TUDelft/9_October_2008 9] | [http://2008.igem.org/TUDelft/10_October_2008 10] | [http://2008.igem.org/wiki/index.php?title=TUDelft/11_October_2008&action=edit 11] | [http://2008.igem.org/wiki/index.php?title=TUDelft/12_October_2008&action=edit 12] |
| [http://2008.igem.org/TUDelft/13_October_2008 13] | [http://2008.igem.org/TUDelft/14_October_2008 14] | [http://2008.igem.org/TUDelft/15_October_2008 15] | [http://2008.igem.org/TUDelft/16_October_2008 16] | [http://2008.igem.org/TUDelft/17_October_2008 17] | [http://2008.igem.org/wiki/index.php?title=TUDelft/18_October_2008&action=edit 18] | [http://2008.igem.org/wiki/index.php?title=TUDelft/19_October_2008&action=edit 19] |
| [http://2008.igem.org/TUDelft/20_October_2008 20] | [http://2008.igem.org/TUDelft/21_October_2008 21] | [http://2008.igem.org/TUDelft/22_October_2008 22] | [http://2008.igem.org/TUDelft/23_October_2008 23] | [http://2008.igem.org/TUDelft/24_October_2008 24] | [http://2008.igem.org/wiki/index.php?title=TUDelft/25_October_2008&action=edit 25] | [http://2008.igem.org/wiki/index.php?title=TUDelft/26_October_2008&action=edit 26] |
| [http://2008.igem.org/wiki/index.php?title=TUDelft/27_October_2008&action=edit 27] | [http://2008.igem.org/wiki/index.php?title=TUDelft/28_October_2008&action=edit 28] | [http://2008.igem.org/wiki/index.php?title=TUDelft/29_October_2008&action=edit 29] | [http://2008.igem.org/wiki/index.php?title=TUDelft/30_October_2008&action=edit 30] | [http://2008.igem.org/wiki/index.php?title=TUDelft/31_October_2008&action=edit 31] | ||
October 24th
Sending parts
Today we sent our lyophilized DNA to MIT. We also sent a number of strains to Baseclear (NL) for sequencing.
Protein Precipitation
Including yesterday's PCA precipitation, four different protein precipitation protocols were conducted to obtain protein content measurements without the disturbance of lysis buffer. The four protocols can be found here, they are PerChloric Acid (PCA), TriChloroAcetic acid (TCA)/acetone, TCA/Deoxycholate (DOC) and methanol/chloroform precipitation. For each of these precipitation methods four samples of constructs BBa_K115012, BBa_K115035 and BBa_K115036 and the calibration curve (bovine serum albumin in H2O) were resuspended in 1x lysis buffer. After precipitation protocol and BCA assay was performed, standard calibration curves were made for all four precipitation protocols together with an untreated calibration curve for comparative reasons. The result can be seen in figure 1.
It is clear from figure 1 that the PCA and acetone/TCA calibration curves are really bad. This can be due to incomplete removal of the lysis buffer and/or differences in precipitation efficiency from sample to sample. The methanol/chloroform standard curve is already much better, but still not good enough for accurate experimentation. The TCA/DOC standard curve can be considered all right. However, a lot of the construct samples measured in this experiment for all precipitation methods gave back negative protein contents. A reason for this could be that protein precipitation in complex (cell extracts) samples takes longer than for simple (calibration curve) samples. During the experiments the shortest incubation periods possible were taken, this could be the reason we did not measure protein content. These protocols won't be used.
