Team:Grinnell/Notebook/Gels

From 2011.igem.org

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<h1>Gel Pics</h1>
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<h2>2011.06.12-2011.06.18</h2>
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<h2>2011.06.26-2011.07.02</h2>
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<h2>Week 5</h2>
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Revision as of 16:40, 5 July 2011

Grinnell Menubar

Gel Pics

2011.06.05-2011.06.11

20110606_PCRproduct 20110610PlamidGel
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

20110614_PCR135 20110614_PCR246 20110614_Promoters 20110616_promotorGel
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.
20110616_promoterPostCleanup 20110617_Pro-esp-stop-rsaA 20110617_rsaA%26esp
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

20110620_promotorsTransformationGel 20110621 BBaGel1 20110621 espAndCombo 20110621 Promoter-esp-Stop-rsaA-pMR10
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.
20110621 rsaA 20110622_PromotersGel2 20110623_esp%2BrsaA_135 20110623_esp%2BrsaA_246
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.
20110624_PrsaA_esp_rsaA 20110624_Pxyl_esp_rsaA 20110624_BBa_K081005_esp_rsaA
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

20110627_esprsaA%2Bpromoters_1 20110627_esprsaA%2Bpromoters_2 20110628_TestDigest 20110629_BBa_K081005
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.
20110629_PromoterInserts1 20110629_PromoterInserts_PrsaA 20110629_PromoterInserts_Both 20110630_espORcombo
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.
20110701_combo1 20110701_combo2
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

20110704_combo
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.

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