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Team:Grinnell/Notebook/Gels
From 2011.igem.org
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<a name='20110704_combo' href='https://2011.igem.org/File:20110704_combo.jpg'> | <a name='20110704_combo' href='https://2011.igem.org/File:20110704_combo.jpg'> | ||
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PCR test of transformation products of insertion of gel extracted DNA into plasmid containing another gene. Lane 1: ladder; lanes 2-4: insertion of <i>rsaA</i> C-terminal into plasmid containing <i>esp</i>; lanes 5-7: insertion of <i>esp</i> into plasmid containing <i>rsaA</i> C-terminal. Smearing of bands is inconclusive and suggests that there may be no DNA (colonies are contaminants) or that freeze-thaw did not succeed in providing sufficient template DNA for PCR. | PCR test of transformation products of insertion of gel extracted DNA into plasmid containing another gene. Lane 1: ladder; lanes 2-4: insertion of <i>rsaA</i> C-terminal into plasmid containing <i>esp</i>; lanes 5-7: insertion of <i>esp</i> into plasmid containing <i>rsaA</i> C-terminal. Smearing of bands is inconclusive and suggests that there may be no DNA (colonies are contaminants) or that freeze-thaw did not succeed in providing sufficient template DNA for PCR. | ||
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| + | Digested miniprep samples of overnight cultures of transformation product that may contain <i>esp</i> and <i>rsaA</i>. Lane 1: <i>esp</i> standard; lanes 2,3: <i>rsaA</i> from gel extraction inserted into pSB1C3 containing <i>esp</i> from plates spread with 20μL of transformation cells; lanes 4,5: <i>rsaA</i> from gel extraction inserted into pSB1C3 containing <i>esp</i> from plates spread with 200μL of transformation cells; lanes 6,7: <i>esp</i> from gel extraction inserted into pSB1C3 containing <i>rsaA</i> from plates spread with 20μL of transformation cells; lanes 8,9: <i>esp</i> from gel extraction inserted into pSB1C3 containing <i>rsaA</i> from plates spread with 200μL of transformation cells; lane 10: ladder. | ||
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| + | Lane 1: ladder; lane 2: digest of miniprep of transformation product of insert of <i>esp</i> from gel extraction into plasmid containing <i>rsaA</i>; lanes 3,4: ligations gel extracted <i>esp</i> and <i>rsaA</i> and plasmid containing the other gene; lane 5: ligation of gel extracted <i>esp</i> with <i>rsaA</i>. Lane 2 shows that ligation was unsuccessful. The rest of the lanes are inconclusive and a follow up gel with additional DNA is to be run. | ||
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| + | Lane 1: ladder; lane 2: ligation of <i>esp</i> and <i>rsaA</i> C-terminal fragments from gel extraction. No <i>rsaA</i> appears to be present, nor any ligation, only <i>esp</i>. | ||
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Revision as of 22:59, 5 July 2011
Gel Pics
2011.06.05-2011.06.11
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| 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. | 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. |
2011.06.12-2011.06.18
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| 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. | 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. | 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. | PCR products of Caulobacter promoters PrsaA and Pxyl. Lane 1: ladder; lane 2: PrsaA; lane 3: Pxyl. |
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| Gel confirming that clean up did not remove DNA fragments. Lane 1: ladder; lane 2: PrsaA; lane 3: Pxyl. | 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. | 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. |
2011.06.19-2011.06.25
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| 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. | 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. | 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. | 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. |
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| 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. | 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. | 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. | 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. |
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| 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. | 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. | 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. |
2011.06.26-2011.07.02
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| 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 Pxyl. While the BBa_K081005 plasmids seem to have some insert, none of these show an insert of appropriate size (~1.3-1.5kb). | 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 PrsaA; lane 8: standard PrsaA with no insert; lane 9: standard Pxyl with no insert. The first lane shows odd results, but none of these seems to have the desired insert. | 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. | 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. |
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| Gel 1 of 3. PCR of transformation cells that should contain an insert of Pxyl 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. | Gel 2 of 3. PCR of transformation cells that should contain an insert of PrsaA 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. | Gel 3 of 3. PCR product of transformation colonies that should contain an insert of either Pxyl or PrsaA into pSB1C3 containing esp and rsaA C-terminal. Lane 1: ladder; lanes 2,3: Pxyl insert PCR using plasmid primers VF2 and VR; lanes 4,5: PrsaA insert PCR using plasmid primers VF2 and VR; lanes 6,7: Pxyl insert PCR using promoter specific forward primer and VR; lanes 8,9: PrsaA insert PCR using promoter specific forward primer and VR. Lanes 8 and 9 suggest a successful insertion of PrsaA, however all of the bands are too small. | 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. |
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| Gel 1 of 2. PCR of transformation products of rsaA C-terminal insert into pSB1C3 containing esp. Lane 1: ladder; lanes 2-7: ligation 1, colonies 1-6; lanes 8-10: ligation 2, colonies 1-3. The complete lack of DNA fragments here means that these ligations failed. | Gel 2 of 2. PCR of transformation products of rsaA C-terminal insert into pSB1C3 containing esp. Lane 1: ladder; lanes 2-4: ligation 2, colonies 4-6; lanes 5-10: ligation 3, colonies 1-6. All the bands here are correct for esp, but not the combination. |
2011.07.03-2011.07.09
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| PCR test of transformation products of insertion of gel extracted DNA into plasmid containing another gene. Lane 1: ladder; lanes 2-4: insertion of rsaA C-terminal into plasmid containing esp; lanes 5-7: insertion of esp into plasmid containing rsaA C-terminal. Smearing of bands is inconclusive and suggests that there may be no DNA (colonies are contaminants) or that freeze-thaw did not succeed in providing sufficient template DNA for PCR. | Digested miniprep samples of overnight cultures of transformation product that may contain esp and rsaA. Lane 1: esp standard; lanes 2,3: rsaA from gel extraction inserted into pSB1C3 containing esp from plates spread with 20μL of transformation cells; lanes 4,5: rsaA from gel extraction inserted into pSB1C3 containing esp from plates spread with 200μL of transformation cells; lanes 6,7: esp from gel extraction inserted into pSB1C3 containing rsaA from plates spread with 20μL of transformation cells; lanes 8,9: esp from gel extraction inserted into pSB1C3 containing rsaA from plates spread with 200μL of transformation cells; lane 10: ladder. | Lane 1: ladder; lane 2: digest of miniprep of transformation product of insert of esp from gel extraction into plasmid containing rsaA; lanes 3,4: ligations gel extracted esp and rsaA and plasmid containing the other gene; lane 5: ligation of gel extracted esp with rsaA. Lane 2 shows that ligation was unsuccessful. The rest of the lanes are inconclusive and a follow up gel with additional DNA is to be run. | Lane 1: ladder; lane 2: ligation of esp and rsaA C-terminal fragments from gel extraction. No rsaA appears to be present, nor any ligation, only esp. |
