|
|
| (One intermediate revision not shown) |
| Line 293: |
Line 293: |
| | <p> <b>27-08-2012 to 02-09-2012</b> </p> | | <p> <b>27-08-2012 to 02-09-2012</b> </p> |
| | | | |
| - | <p>DNA purification from FFT liquid cultures were made.<br/></br> | + | <p> |
| - | Nanodrop was performed on it:<br/>
| + | After 48 hours in the incubator, the 1-FFT colonies finaly reached a high enough concentration for sequencing.<br/><br/> |
| - | 1: 83,6 ηg/μL<br/>
| + | |
| - | 2: 17,2 ηg/μL<br/>
| + | |
| - | 3: 121,1 ηg/μL<br/>
| + | |
| - | 4: 48,1 ηg/μL<br/>
| + | |
| - | 5: 99,1 ηg/μL<br/>
| + | |
| - | 6: 59,6 ηg/μL<br/>
| + | |
| - | 7: nothing(an error must have occurred)<br/>
| + | |
| - | 8: 59,6 ηg/μL<br/></br>
| + | |
| - | We made check-digest with EcoRI+SpeI to examine the plasmids in preparation of mutagenesis.
| + | |
| - | The check digest showed that some had the appropriate digested sizes we would expect from a plasmid of 3kb and a gene of 2kb.<br/></br>
| + | |
| - | Mutagenesis was run on these FFT colonies containing all primers.<br/>
| + | |
| - | We made new SST cultures again and plated them on AMP-resistance plates. <br/>
| + | |
| - | Isolated FFT and SST plasmids and sent them for sequencing.<br/>
| + | |
| - | Afterwards we did a digest and transformation on FFT and plated them.<br/>
| + | |
| - | We made liquid cultures of FFT and SST and incubated them over night. Later we made transformation on the three parts we chose from the iGEM kit.<br/>
| + | |
| - | 1) R0010 <br/>
| + | |
| - | 2) E0040<br/>
| + | |
| - | 3) B0015<br/>
| + | |
| - | Afterwards we plated them on AMP-resistance plates. <br/>
| + | |
| - | We made liquid cultures of the three parts<br/></br>
| + | |
| - | <h2>Purification of SST, FFT, B0015, E0040 and R0010 and following PCR</h2></br>
| + | |
| - | <p>The O.N. cultures of SST, FFT and our 3 parts (B0015/terminator, E0040/GFP and R0010/LacL) showed promising results of growth. A miniprep was made from the 32 samples and a Nanodrop as well which gave the following results:
| + | |
| - | </p>
| + | |
| | | | |
| - | <TABLE border="1" width="100%">
| + | Meanwhile, all the SST colonies had died out. To avoid unnecesary excess work, we simply ran PCR with all mutagenesis primers on an older 1-SST containing pJET plasmid. Miraculous, both gel band length and check digest on the resulting sequence yielded perfect results. Why had we not done this before?<br/><br/> |
| - | <CAPTION><EM>NanoDrop Results</EM></CAPTION>
| + | |
| - | <TR><TH>1. R0010, culture 1 <TD>18,0 ng/ul<TH>17. R0010, culture 6 <TD>28,1 ng/ul
| + | |
| - | <TR><TH>2. B0015, culture 3<TD>67,9 ng/ul<TH>18. SST plate 2, culture 3 <TD>48,1 ng/ul
| + | |
| - | <TR><TH>3. B0015, culture 5 <TD>62,9 ng/ul <TH>19. B0015, culture 6 <TD>59,0 ng/ul
| + | |
| - | <TR><TH>4. R0010, culture 3 <TD>70,5 ng/ul <TH>20. B0015, culture 2 <TD>67,8 ng/ul
| + | |
| - | <TR><TH>5. R0010, culture 5 <TD>69,2 ng/ul <TH>21. E0040, culture 4 <TD>43,6 ng/ul
| + | |
| - | <TR><TH>6. E0040, culture 6 <TD>35,5 ng/ul <TH>22. FFT plate 8, culture 1 <TD>7,7 ng/ul
| + | |
| - | <TR><TH>7. E0040, culture 1 <TD>66,2 ng/ul <TH>23. FFT plate 8, culture 2 <TD>7,0 ng/ul
| + | |
| - | <TR><TH>8. E0040, culture 3 <TD>47,5 ng/ul <TH>24. R0010, culture 5 <TD>24,6 ng/ul
| + | |
| - | <TR><TH>9. SST plate 1, culture 1 <TD>13,9 ng/ul <TH>25. FFT plate 4, culture 2 <TD>6,9 ng/ul
| + | |
| - | <TR><TH>10. R0010, culture 2 <TD>22,3 ng/ul <TH>26. FFT plate 4, culture 3 <TD>7,9 ng/ul
| + | |
| - | <TR><TH>11. E0040, culture 5 <TD>98,9 ng/ul <TH>27. FFT plate 4, culture 5 <TD>5,6 ng/ul
| + | |
| - | <TR><TH>12. FFT plate 8, culture 5 <TD>22,4 ng/ul <TH>28. FFT plate 6, culture 6 <TD>7,3 ng/ul
| + | |
| - | <TR><TH>13. FFT plate 4, culture 3 <TD>11,5 ng/ul <TH>29. FFT plate 4, culture 1 <TD>6,9 ng/ul
| + | |
| - | <TR><TH>14. E0040, culture 2 <TD>50,2 ng/ul <TH>30. B0015, culture 4 <TD>57,3 ng/ul
| + | |
| - | <TR><TH>15. B0015, culture 7 <TD>29,0 ng/ul <TH>31. FFT plate 8, culture 7 <TD>6,1 ng/ul
| + | |
| - | <TR><TH>16. FFT plate 2, culture 1 <TD>102,9 ng/ul <TH>32. SST plate 2, culture 1 <TD>75,9 ng/ul
| + | |
| - | | + | |
| - | </TABLE>
| + | |
| - | | + | |
| - | <p>New medium was added to the liquid cultures and stored in the incubator at 37C for future use.
| + | |
| - | 16 samples from the miniprep was picked out: 12, 13, 18, 22, 23, 25, 26, 27, 28, 29, 31, 32 plus 4 with GFP insert and a PCR with a proofreading polymerase (Phusion Hot Star II) was run.</br>
| + | |
| - | We got no useable results from this. There were a variation of lengths, and none of the ones we would expect.<br/>
| + | |
| - | Then we did PCR on the two genes FFT and SST with the extension primers at 71°C annealing temperature. We ran a gel on the PCR product, but only had results for FFT. Therefore we purified the FFT genes. Another PCR was made for SST and it was thereafter ligated into R0010 and transformed for overnight incubation. </br>
| + | |
| - | | + | |
| - | | + | |
| - | <h2>Transformation of TOP10 competent E. coli with GFP and terminator</h2>
| + | |
| - | <p>The GFP+terminater sequence was transformed and plated out on agar plates. They didn’t show any colonies.</br> | + | |
| - | For PCR of sequences from mini prep:</br>
| + | |
| - | FFT forward primer: FFT1-shine-for, number: 23, Tm: 69</br>
| + | |
| - | FFT Reverse primer: FFT1-ext-rev, number: 4, Tm: 67</br>
| + | |
| - | </br>
| + | |
| - | SST forward primer: SST1-shine-for, number 22, Tm: 71</br>
| + | |
| - | SST reverse primer: FFT1-ext-rev, number 8, Tm: 66</br>
| + | |
| - | </br>
| + | |
| - | We transformed the RFP/terminator part into TOP10 with a new transformation protocol, where the effectivity was tested after 30 or 90 seconds heat shock.</br>
| + | |
| - | We made a PCR on everything and all of our parts and genes with VF2 and VR primers. Got very bad results.
| + | |
| - |
| + | |
| | | | |
| | + | In the following days 1-SST ligated into pJET, grown on ampicillin plates, transformed into Top10, transfered to liquid medium and miniprepped for sequencing.<br/> |
| | + | </p> |
| | | | |
| | | | |
"
