|
|
| (20 intermediate revisions not shown) |
| Line 1: |
Line 1: |
| | {{:Team:Grinnell/Template/Home}} | | {{:Team:Grinnell/Template/Home}} |
| - | | + | <html> |
| - | ==Gel Pictures==
| + | <body> |
| - | | + | <h1>Gel Pics</h1> |
| - | ===June 6 to 10===
| + | <div style="float:right; position:relative; left:-125px; top:-5px; z-index:-1"> |
| - | <gallery> | + | <img alt="Our first gel" src='https://static.igem.org/mediawiki/2011/3/32/20110606_PCRproduct.jpg' height=200px /> |
| - | <html><a name="1"></a></html>File:20110606_PCRproduct.jpg|PCR products on DNA gel. Lane 1: ladder; Lane 2: ''rsaA'' from liquid culture ''Caulobacter''; Lane 3: ''rsaA'' from plate culture ''Caulobacter''; Lane 4: ''esp'' from ''S. epidermidis''. | + | </div> |
| - | <html><a name="2"></a></html>File:20110610PlamidGel.jpg|Gel results for transformation of ligations. Lane 1: ladder; Lane 2: digested plasmid with ''rsaA'' C-terminal; Lane 3: digested plasmid with ''rsaA'' C-terminal and ''esp''; Lanes 4-8: digested plasmids from various colonies with ''esp''. | + | <p>We collected more gel data than we could fit on one page. Please go on to one of the following pages:</p> |
| - | </gallery>
| + | <ul> |
| - | | + | < li><a href=" https:// 2011. igem. org/ Team: Grinnell/ Notebook/ Gels/ DspB"> DspB</a></ li> |
| - | ===June 13 to 17===
| + | < li><a href=" https:// 2011. igem. org/ Team: Grinnell/ Notebook/ Gels/ Esp"> Esp</a></ li> |
| - | <gallery>
| + | < li><a href=" https:// 2011. igem. org/ Team: Grinnell/ Notebook/ Gels/ Promoters"> Promoters</a></ li> |
| - | <html><a name=""></a></html>File:20110614_PCR135.jpg|Gel 1 of 2. Lane 1: ladder; lanes 2-4: PCR product using VF2 and VR of plasmid containing ''esp'' ligated with ''rsaA'' C-terminal that had been digested 20110606; lane 5: PCR product of plasmid containing ''esp''; lanes 6-8: PCR product using VF2 and VR of plasmid containing ''esp'' ligated with ''rsaA'' C-terminal PCR product from 20110605. Only lane 6 shows successful ligation of ''esp'' and ''rsaA'' C-terminal.
| + | </ul> |
| - | <html><a name=""></a></html>File:20110614_PCR246.jpg|Gel 2 of 2. Lane 1: ladder; lanes 2-4: PCR product using VF2 and VR of plasmid containing ''esp'' ligated with ''rsaA'' C-terminal that had been digested 20110606; lane 5: PCR product of plasmid containing ''esp''; lanes 6-8: PCR product using VF2 and VR of plasmid containing ''esp'' ligated with ''rsaA'' C-terminal PCR product from 20110605. None of these show successful ligation.
| + | </body> |
| - | <html><a name=""></a></html>File:20110614_Promoters.jpg|Gel of PCR of ''Caulobacter'' promoters Pxyl (inducible) and PrsaA (constitutive). Lane 1: ladder; lane 2: PrsaA; Lane 3: Pxyl. Bands appear, but are hazy and spread out due to the small size of DNA fragments. | + | </html> |
| - | <html><a name=""></a></html>File:20110616_promotorGel.jpg|PCR products of ''Caulobacter'' promoters PrsaA and Pxyl. Lane 1: ladder; lane 2: PrsaA; lane 3: Pxyl.
| + | |
| - | <html><a name=""></a></html>File:20110616_promoterPostCleanup.jpg|Gel confirming that clean up did not remove DNA fragments. Lane 1: ladder; lane 2: PrsaA; lane 3: Pxyl.
| + | |
| - | <html><a name=""></a></html>File:20110617_Pro-esp-stop-rsaA.jpg|Gel of PCR products of transformations of insertion of Biobrick promoter BBa_K081005 into pSB1C3 containing esp and rsaA C-terminal. Lane 1: ladder; lanes 2-4 and 6-8: PCR product of transformation survivors; lane 5: control DNA of ''esp'' + ''rsaA'' C-terminal. Only lane 2 shows something that may be success, but as the fragment for the promoter is so small, it is difficult to confirm based solely on gel electrophoresis.
| + | |
| - | <html><a name=""></a></html>File:20110617_rsaA%26esp.jpg|Gel of PCR product of ''esp'' and ''rsaA'' C-terminal using new primers. Lane 1: ladder; lanes 2,3: PCR product of ''rsaA'' from chromosomal ''Caulobacter'' DNA; lane 4: PCR product of ''esp'' using new primers from chromosomal ''S. epidermidis'' DNA.
| + | |
| - | </gallery> | + | |
| - | | + | |
| - | ===June 20 to 24===
| + | |
| - | <gallery> | + | |
| - | <html><a name=""></a></html>File:20110620_promotorsTransformationGel.jpg|Gel of Plasmid PCR of promoters. Presence of two small bands shows contamination of samples. Lane 1: ladder; lanes 2-5: plasmid PCR of transformation survivors.
| + | |
| - | <html><a name=""></a></html>File:20110621_BBaGel1.jpg|Gel of BBa_K081005 digests from minipreps. Lane 1: ladder; lane 2: digest of miniprep from overnights that should carry the desired promoter insert. Digest shows one band: the linearized plasmid.
| + | |
| - | <html><a name=""></a></html>File:20110621_espAndCombo.jpg|Lanes 1-6: plasmid PCR of transformed cells that should carry the desired ''esp'' insert; lane 7: ladder; lanes 8-10: plasmid PCR of transformed cells that should be carrying ''esp'' + ''rsaA'' C-terminal insert. A few of the ''esp'' inserts succeeded, but as expected the three piece ligation failed again.
| + | |
| - | <html><a name=""></a></html>File:20110621_Promoter-esp-Stop-rsaA-pMR10.jpg|Lanes 1-3 and 5-8: plasmid PCR of transformed cells that should carry the Promoter-esp-stop-rsaA insert in plasmid pMR10; lane 4: ladder. Only lane 3 shows any success.
| + | |
| - | <html><a name=""></a></html>File:20110621_rsaA.jpg|Lanes 1-3 and 5-7: plasmid PCR of colonies that should be carrying ''rsaA'' C-terminal insert in pSB1C3; lane 4: ladder. Lanes 3 and 4 seem to show insert at significant concentrations. | + | |
| - | < html><a name=" "></a></html>File: 20110622_PromotersGel2.jpg|Lane 1: ladder; lanes 2-4: plasmid PCR product of various promoter insertions into pSB1C3. Smearing due to leftover circular plasmid. Insertions seem to have been successful. | + | |
| - | <html><a name=""></a></html>File:20110623_esp%2BrsaA_135.jpg|Gel 1 of 2. Colony PCR of transformation products of insertions of ''esp'' from PCR into pSB1C3 containing ''rsaA'' C-terminal, insertions of ''rsaA'' C-terminal into pSB1C3 containing ''esp'', and ''esp'' and ''rsaA'' C-terminal digests from pSB1C3 into pMR10 as a three piece ligation. Lane 1: ladder; lanes 2-4: ''esp'' from PCR inserted into ''rsaA'' containing pSB1C3; lanes 5-7: ''rsaA'' from PCR inserted into ''esp'' containing pSB1C3; lanes 8-10: ''esp'' and ''rsaA'' digests from plasmid insertion into pMR10. Lanes 2-4 show correct insertion length, but none of the other fragments are the correct length.
| + | |
| - | <html><a name=""></a></html>File:20110623_esp%2BrsaA_246.jpg|Gel 2 of 2. Colony PCR of transformation products of insertions of ''esp'' from PCR into pSB1C3 containing ''rsaA'' C-terminal, insertions of ''rsaA'' C-terminal into pSB1C3 containing ''esp'', and ''esp'' and ''rsaA'' C-terminal digests from pSB1C3 into pMR10 as a three piece ligation. Lane 1: ladder; lanes 2-4: ''esp'' from PCR inserted into ''rsaA'' containing pSB1C3; lanes 5-7: ''rsaA'' from PCR inserted into ''esp'' containing pSB1C3; lanes 8-10: ''esp'' and ''rsaA'' digests from plasmid insertion into pMR10. Lanes 2-4 show correct insertion length, but none of the other fragments are the correct length.
| + | |
| - | <html><a name=""></a></html>File:20110624_PrsaA_esp_rsaA.jpg|Colony PCR products of transformation products of insertions of ''esp'' + ''rsaA'' C-terminal into plasmid containing ''Caulobacter'' constitutive promoter PrsaA. Lane 1: ladder; lanes 2-7: PCR products; lane 8: standard PCR product of pSB1C3 containing only PrsaA. Transformation was successful, but ligation, and perhaps digestion, were not.
| + | |
| - | <html><a name=""></a></html>File:20110624_Pxyl_esp_rsaA.jpg|Colony PCR products of transformation products of insertion of ''esp'' + ''rsaA'' into pSB1C3 containing ''Caulobacter'' inducible promoter Pxyl (induced in presence of xylene). Lane 1: ladder; lanes 2-7: PCR products; lane 8: standard PCR product of pSB1C3 containing only Pxyl. Transformation was successful, but ligation was not.
| + | |
| - | <html><a name=""></a></html>File:20110624_BBa_K081005_esp_rsaA.jpg|Colony PCR amplified DNA fragments of transformation products of inserts into plasmid containing existing Biobrick promoter BBa_K081005. Lane 1: ladder; lanes 2-7: PCR products; lane 8: standard DNA fragment identified as containing BBa_K081005. The lack of DNA may be due to a breakdown in our antibiotic; the transformation is being repeated.
| + | |
| - | </gallery> | + | |
| - | | + | |
| - | ===June 27 to August 1===
| + | |
| - | | + | |
| - | <gallery>
| + | |
| - | <html><a name=" "></a></html>File: 20110627_esprsaA%2Bpromoters_1.jpg|Gel 1 of 2. PCR products of transformation cells that should contain ''esp'' + ''rsaA'' C-terminal insert into plasmids containing various promoters. Lane 1: ladder; lanes 2-5: inserts into plasmid containing BBa_K081005; lane 6: standard BBa_K081005 containing no insert; lanes 7-10: inserts into plasmid containing P<html><sub>xyl</ sub></ html>. While the BBa_K081005 plasmids seem to have some insert, none of these show an insert of appropriate size (~1. 3-1.5kb). | + | |
| - | <html><a name=""></a></html>File:20110627_esprsaA%2Bpromoters_2.jpg|Gel 2 of 2. PCR products of transformation cells that should contain ''esp'' + ''rsaA'' C-terminal insert into plasmids containing various promoters. Lane 1: ladder; lanes 2-7: inserts into plasmid containing P<html><sub>rsaA</sub>; lane 8: standard P<sub>rsaA</sub> with no insert; lane 9: standard P<sub>xyl</sub> with no insert.</html> The first lane shows odd results, but none of these seems to have the desired insert.
| + | |
| - | <html><a name=""></a></html>File:20110628_TestDigest.jpg|Test to ensure all of our restriction enzymes are still active. Lane 1: ladder; lane 2: cut with Eco RI; lane 3: cut with XbaI; lane 4: standard uncut DNA fragment for lanes 2 and 3; lane 5: cut with SpeI; lane 6: cut with PstI; lane 7: standard uncut DNA fragment for lanes 5 and 6. There is an apparent decrease in size from standard DNA fragments to the digested samples. In the digest lanes there is also a dim band fairly far down the gel corresponding to the small end piece that is the other product of digestion.
| + | |
| - | < html><a name=" "></a></html>File: 20110629_BBa_K081005.jpg|Lane 1: ladder; lane 2: colony PCR product for promoter BBa_K081005 in preparation for digestion and insertion into plasmid containing ''esp'' and ''rsaA'' C-terminal. Result is positive. | + | |
| - | <html><a name=""></a></html>File:20110629 PromoterInserts1.jpg| Gel 1 of 3. PCR of transformation cells that should contain an insert of P<html><sub>xyl</sub></html> into pSB1C3 containing ''esp'' and ''rsaA'' C-terminal. Lane 1: ladder; lanes 2-5: PCR with plasmid primers VF2 and VR; lanes 6-9: PCR with promoter specific forward primer and plasmid reverse primer VR. The lack of DNA is disturbing and suggests that our plates are ineffective or contaminated.
| + | |
| - | <html><a name=""></a></html>File:20110629_PromoterInserts_PrsaA.jpg|Gel 2 of 3. PCR of transformation cells that should contain an insert of P<html><sub>rsaA</sub></html> into pSB1C3 containing ''esp'' and ''rsaA'' C-terminal. Lane 1: ladder; lanes 2-5: PCR with plasmid primers VF2 and VR; lanes 6-9: PCR with promoter specific forward primer and plasmid reverse primer VR. Bands in lanes 2, 3, and 4 suggests that these colonies at least contained the plasmid, but the apparent size of the band is too small for ''esp'' and ''rsaA'' C-terminal together; rather it is the correct size for just ''rsaA'' C-terminal.
| + | |
| - | <html><a name=""></a></html>File:20110629_PromoterInserts_Both.jpg|Gel 3 of 3. PCR product of transformation colonies that should contain an insert of either P<html><sub>xyl</sub> or P<sub>rsaA</sub></html> into pSB1C3 containing ''esp'' and ''rsaA'' C-terminal. Lane 1: ladder; lanes 2,3: P<html><sub>xyl</sub> insert PCR using plasmid primers VF2 and VR; lanes 4,5: P<sub>rsaA</sub> insert PCR using plasmid primers VF2 and VR; lanes 6,7: P<sub>xyl</sub> insert PCR using promoter specific forward primer and VR; lanes 8,9: P<sub>rsaA</sub> insert PCR using promoter specific forward primer and VR. Lanes 8 and 9 suggest a successful insertion of P<sub>rsaA</sub></html>, however all of the bands are too small.
| + | |
| - | <html><a name=""></a></html>File:20110630 espORcombo.jpg|Verification of presence of lack of ''rsaA'' C-terminal insert in pSB1C3 containing ''esp''. Lane 1: ladder; lane 2: digested PCR of possible insert; lane 3: standard digested ''esp''. The band is faint, but there appears to be a band at the same height as the standard as well as a couple higher bands for leftover plasmids, suggesting that there was no insertion of ''rsaA'' C-terminal.
| + | |
| - | | + | |
| - | </gallery>
| + | |
We collected more gel data than we could fit on one page. Please go on to one of the following pages:
"
