Team:Grinnell/Notebook/Gels

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''.

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''.

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.

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.

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.

File:20110616_promotorGel.jpg|PCR products of ''Caulobacter'' promoters PrsaA and Pxyl.  Lane 1: ladder; lane 2: PrsaA; lane 3: Pxyl.

File:20110616_promoterPostCleanup.jpg|Gel confirming that clean up did not remove DNA fragments. Lane 1: ladder; lane 2: PrsaA; lane 3: Pxyl.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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>_xyl</html>.  While the BBa_K081005 plasmids seem to have some insert, none of these show an insert of appropriate size (~1.3-1.5kb).

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>_rsaA; lane 8: standard P_rsaA with no insert; lane 9: standard P_xyl with no insert.</html> The first lane shows odd results, but none of these seems to have the desired insert.

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.

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.

File:20110629 PromoterInserts1.jpg| Gel 1 of 3. PCR of transformation cells that should contain an insert of P<html>_xyl</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.

File:20110629_PromoterInserts_PrsaA.jpg|Gel 2 of 3. PCR of transformation cells that should contain an insert of P<html>_rsaA</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.

File:20110629_PromoterInserts_Both.jpg|Gel 3 of 3. PCR product of transformation colonies that should contain an insert of either P<html>_xyl or P_rsaA</html> into pSB1C3 containing ''esp'' and ''rsaA'' C-terminal. Lane 1: ladder; lanes 2,3: P<html>_xyl insert PCR using plasmid primers VF2 and VR; lanes 4,5: P_rsaA insert PCR using plasmid primers VF2 and VR; lanes 6,7: P_xyl insert PCR using promoter specific forward primer and VR; lanes 8,9: P_rsaA insert PCR using promoter specific forward primer and VR. Lanes 8 and 9 suggest a successful insertion of P_rsaA</html>, however all of the bands are too small.

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.