IGEM:Harvard/2007/Gels: Difference between revisions
No edit summary |
|||
| (34 intermediate revisions by 5 users not shown) | |||
| Line 1: | Line 1: | ||
[[Image: Week 0 Plasmid Gel.jpg|thumb|left|250px]] | == Week 0 gel == | ||
[[Image: Week 0 Plasmid Gel.jpg|thumb|left|250px]]<br> | |||
Description: Week 0 gel<br> | |||
Description: Week 0 gel | |||
Well 1 Ladder <br> | Well 1 Ladder <br> | ||
Well 2 OmpA1 in PET <br> | Well 2 OmpA1 in PET <br> | ||
| Line 9: | Line 9: | ||
Well 5 OmpA1 +strep tag in PET <br> | Well 5 OmpA1 +strep tag in PET <br> | ||
Well 6 OmpA2 +his tag in PET <br> | Well 6 OmpA2 +his tag in PET <br> | ||
Well 7 OmpA2 +strep tag in PET <br> | Well 7 OmpA2 +strep tag in PET <br><br><br><br> | ||
[[Image:June 19 Gel.jpg|thumb|left|250px]] | == 6/20 gel == | ||
[[Image:June 19 Gel.jpg|thumb|left|250px]] | |||
Description: 6/20 gel<br> | Description: 6/20 gel<br> | ||
Well 1 Ladder (10 kb) | Well 1 Ladder (10 kb) <br> | ||
Well 2 OmpA1, Nhe1, Pst1, Group 1 27.5 uL<br> | |||
Well 2 OmpA1, Nhe1, Pst1, Group 1 <br> | Well 3 OmpA1, Nhe1, Pst1, Group 1 27.5 uL<br> | ||
Well 3 OmpA1, Nhe1, Pst1, Group 1 <br> | Well 4 OmpA1, Nhe1, Pst1, Group 2 27.5 uL<br> | ||
Well 4 OmpA1, Nhe1, Pst1, Group 2 <br> | Well 5 OmpA1, Nhe1, Pst1, Group 2 27.5 uL<br> | ||
Well 5 OmpA1, Nhe1, Pst1, Group 2 <br> | Well 6 OmpA1, Nhe1, Pst1, Group 2 27.5 uL<br> | ||
Well 6 OmpA1, Nhe1, Pst1, Group 2 <br> | Well 7 OmpA2, Nhe1, Pst1, Group 4 27.5 uL<br> | ||
Well 7 OmpA2, Nhe1, Pst1, Group 4 <br> | Well 8 OmpA2, Nhe1, Pst1, Group 4 27.5 uL<br> | ||
Well 8 OmpA2, Nhe1, Pst1, Group 4 <br> | Well 9 OmpA2, Nhe1, Pst1, Group 3 27.5 uL<br> | ||
Well 9 OmpA2, Nhe1, Pst1, Group 3 <br> | Well 10 OmpA2, Nhe1, Pst1, Perry's 27.5 uL<br> | ||
Well 10 OmpA2, Nhe1, Pst1, Perry's<br> | |||
Well 11 OmpA2, Nhe1, Pst1, Perry's<br> | Well 11 OmpA2, Nhe1, Pst1, Perry's<br> | ||
See Notebook page for gel extraction weights and values.<br> | See Notebook page for gel extraction weights and values.<br> | ||
== 6/19 gel after excision== | |||
[[Image:6-19-07_gel_after_excision.jpg|thumb|left|250px]]<br> | |||
[[Image:6-19-07_gel_after_excision.jpg|thumb|left|250px]] | |||
Description: 6/19 gel after excision <br> | Description: 6/19 gel after excision <br> | ||
| Line 47: | Line 39: | ||
Well 5 OmpA1 +strep tag in PET <br> | Well 5 OmpA1 +strep tag in PET <br> | ||
Well 6 OmpA2 +his tag in PET <br> | Well 6 OmpA2 +his tag in PET <br> | ||
Well 7 OmpA2 +strep tag in PET <br> | Well 7 OmpA2 +strep tag in PET <br><br><br><br> | ||
== 6/22 overexpression gel == | |||
[[Image:6-22-07_overexpression_gel.jpg|thumb|left|250px]] | [[Image:6-22-07_overexpression_gel.jpg|thumb|left|250px]] | ||
Description: 6/22 overexpression gel <br> | <br>Description: 6/22 overexpression gel <br> | ||
lane 1 Ladder <br> | lane 1 Ladder <br> | ||
lane 2 OmpA1 + his6 non-induced<br> | lane 2 OmpA1 + his6 non-induced<br> | ||
| Line 66: | Line 53: | ||
lane 7 OmpA2 + his6 induced <br> | lane 7 OmpA2 + his6 induced <br> | ||
lane 8 OmpA2 + strep2 non-induced <br> | lane 8 OmpA2 + strep2 non-induced <br> | ||
lane 9 OmpA2 + strep2 induced <br> | lane 9 OmpA2 + strep2 induced <br><br> | ||
== 6/22 overexpression gel2, ladder contaminated == | |||
[[Image:6-22-07_overexpressgel2_laddercontaminate.jpg|thumb|left|250px]] | |||
<br><br><br> | |||
Description: Contaminated 6/22 overexpression gel <br> | |||
Explanation: Failed gel because of contamination in each lane by the ladder, which was mistakenly added. | |||
<br><br><br><br><br><br> | |||
== 6/27 plasmid gel, performed by Stephanie == | |||
[[Image:Gelpostpcrplasmid0627.jpg|thumb|left|250px]] | |||
Description: Gel using Lpp+OmpA1+pet29 and LppOmpA2+pet29 | |||
Explanation: | |||
For further information concerning amounts of plasmid in sample, please refer to Stephanie's notebook page (under Stephanie's personal page, shlo/notebook) | |||
Wells are as follows:<br> | |||
Well 1: 10uL ladder of 1kB plus, from Invitrogen | |||
<i> Note: this ladder cannot be used to quantify - perhaps a reason we should just use 1kB ladder, rather than 1kB+, in the future</i><br> | |||
Well 2: 1uL OmpA1plasmid (diluted to 20uL)<br> | |||
Well 3: 3uL OmpA1plasmid (diluted to 20)<br> | |||
Well 4: 1uL OmpA2plasmid (diluted to 20)<br> | |||
Well 5: 3uL OmpA2plasmid (diluted to 20)<br><br> | |||
== 6/27 protein denaturation electrophoresis gel == | |||
[[Image:1_protGel_Jul7.jpg|thumb|left|250px]]<br> | |||
<br>Description: 6/27 denaturation electrophoresis gel <br> | |||
The rightmost lane is the ladder | |||
lane 1 Ladder <br> | |||
lane 2 OmpA1 + strep before induction with IPTG<br> | |||
lane 3 OmpA1 + his6 before induction<br> | |||
lane 4 OmpA1 + strep after induction<br> | |||
lane 5 OmpA1 + his6 after induction<br> | |||
lane 6 OmpA1 + his6 after induction<br> | |||
lane 7 OmpA1 + strep after induction<br> | |||
lane 8 OmpA1 + his6 before induction<br> | |||
lane 9 OmpA1 + strep before induction<br><br> | |||
''Note that there were 2 copies of each sample: samples in lanes 2 to 5 were denatured for 2 minutes as per protocol while samples in lanes 6 to 9 were denatured for a 10 minutes, Sammy S.'' | |||
---- | == 7/05 protein gel, Kevin and Sammy == | ||
[[Image:6- | [[Image: 2007-07-06protGel2.jpg|thumb|left|300px]]<br> | ||
'''Gel 2: induced immediately'''<br> | |||
Lane 1: Ladder<br> | |||
Lane 2: Strep No IPTG<br> | |||
Lane 3: Strep denatured after 30 min<br> | |||
Lane 4: Strep denatured after 60 min<br> | |||
Lane 5: Strep denatured after 90 min<br> | |||
Lane 6: Strep denatured after 120 min <br> | |||
Lane 7: His No IPTG<br> | |||
Lane 8: His denatured after 30 min <br> | |||
Lane 9: His denatured after 60 min <br> | |||
Lane 10: His denatured after 90 min <br><br><br><br> | |||
[[Image: 2007-07-06protGel1.jpg|thumb|left|350px]]<br> | |||
'''Gel 1'''<br> | |||
Lane 1: Ladder<br> | |||
Lane 2: His 0 min induction<br> | |||
Lane 3: His 30 min induction<br> | |||
Lane 4: His 60 min induction<br> | |||
Lane 5: His 90 min induction<br> | |||
Lane 6: His 120 min induction<br> | |||
Lane 7: Strep 0 min induction<br> | |||
Lane 8: Strep 30 min induction<br> | |||
Lane 9: Strep 60 min induction<br> | |||
Lane 10: Strep 90 min induction<br> | |||
Lane 11: Strep 120 min induction<br> | |||
Lane 12: His 0 min induction 120 min denature<br> | |||
== 7/09 Egel, Alex == | |||
[[Image: mutagenesisgel.jpg|thumb|left|300px]]<br> | |||
'''Gel 2: induced immediately'''<br> | |||
Lane 1: Ladder<br> | |||
Lane 2: Undigested FecR (-) R1A<br> | |||
Lane 3: Pst1 Digested Fec R (-) R1A<br> | |||
Lane 4: Undigested FecR (-) R5B<br> | |||
Lane 5: Pst1 Digested Fec R (-) R5B<br> | |||
Lane 6: Pst1 Digested Fec R (+) <br> | |||
Lane 7: Undigested FecA (-) A1A<br> | |||
Lane 8: Pst1 Digested Fec A (-) A1A<br> | |||
Lane 9: Undigested FecR (-) A5B<br> | |||
Lane 10: Pst1 Digested Fec R (-) A5B<br> | |||
Lane 11: Pst1 Digested Fec R (+) A1B<br> | |||
Lane 12: Pst1 Digested Fec A (+) <br> | |||
<br> | |||
== 7/26/07 Egel, Kevin and Mike == | |||
[[Image: KevinMikeGel.jpg|thumb|left|300px]]<br> | |||
'''Gel: AIDA after PCR'''<br> | |||
Lane 1: Ladder<br> | |||
Lane 2: AIDA1+his<br> | |||
Lane 3: AIDA1+strep<br> | |||
Result: We did not get ideal results, though we did get light expression where we expected. | |||
Latest revision as of 07:39, 30 July 2007
Week 0 gel
Description: Week 0 gel
Well 1 Ladder
Well 2 OmpA1 in PET
Well 3 OmpA2 in PET
Well 4 OmpA1 +his tag in PET
Well 5 OmpA1 +strep tag in PET
Well 6 OmpA2 +his tag in PET
Well 7 OmpA2 +strep tag in PET
6/20 gel
Description: 6/20 gel
Well 1 Ladder (10 kb)
Well 2 OmpA1, Nhe1, Pst1, Group 1 27.5 uL
Well 3 OmpA1, Nhe1, Pst1, Group 1 27.5 uL
Well 4 OmpA1, Nhe1, Pst1, Group 2 27.5 uL
Well 5 OmpA1, Nhe1, Pst1, Group 2 27.5 uL
Well 6 OmpA1, Nhe1, Pst1, Group 2 27.5 uL
Well 7 OmpA2, Nhe1, Pst1, Group 4 27.5 uL
Well 8 OmpA2, Nhe1, Pst1, Group 4 27.5 uL
Well 9 OmpA2, Nhe1, Pst1, Group 3 27.5 uL
Well 10 OmpA2, Nhe1, Pst1, Perry's 27.5 uL
Well 11 OmpA2, Nhe1, Pst1, Perry's
See Notebook page for gel extraction weights and values.
6/19 gel after excision
Description: 6/19 gel after excision
Well 1 Ladder
Well 2 OmpA1 in PET
Well 3 OmpA2 in PET
Well 4 OmpA1 +his tag in PET
Well 5 OmpA1 +strep tag in PET
Well 6 OmpA2 +his tag in PET
Well 7 OmpA2 +strep tag in PET
6/22 overexpression gel
Description: 6/22 overexpression gel
lane 1 Ladder
lane 2 OmpA1 + his6 non-induced
lane 3 OmpA1 + his6 induced
lane 4 OmpA1 + strep2 non-induced
lane 5 OmpA1 + strep2 induced
lane 6 OmpA2 + his6 non-induced
lane 7 OmpA2 + his6 induced
lane 8 OmpA2 + strep2 non-induced
lane 9 OmpA2 + strep2 induced
6/22 overexpression gel2, ladder contaminated
Description: Contaminated 6/22 overexpression gel
Explanation: Failed gel because of contamination in each lane by the ladder, which was mistakenly added.
6/27 plasmid gel, performed by Stephanie
Description: Gel using Lpp+OmpA1+pet29 and LppOmpA2+pet29
Explanation:
For further information concerning amounts of plasmid in sample, please refer to Stephanie's notebook page (under Stephanie's personal page, shlo/notebook)
Wells are as follows:
Well 1: 10uL ladder of 1kB plus, from Invitrogen
Note: this ladder cannot be used to quantify - perhaps a reason we should just use 1kB ladder, rather than 1kB+, in the future
Well 2: 1uL OmpA1plasmid (diluted to 20uL)
Well 3: 3uL OmpA1plasmid (diluted to 20)
Well 4: 1uL OmpA2plasmid (diluted to 20)
Well 5: 3uL OmpA2plasmid (diluted to 20)
6/27 protein denaturation electrophoresis gel
Description: 6/27 denaturation electrophoresis gel
The rightmost lane is the ladder
lane 1 Ladder
lane 2 OmpA1 + strep before induction with IPTG
lane 3 OmpA1 + his6 before induction
lane 4 OmpA1 + strep after induction
lane 5 OmpA1 + his6 after induction
lane 6 OmpA1 + his6 after induction
lane 7 OmpA1 + strep after induction
lane 8 OmpA1 + his6 before induction
lane 9 OmpA1 + strep before induction
Note that there were 2 copies of each sample: samples in lanes 2 to 5 were denatured for 2 minutes as per protocol while samples in lanes 6 to 9 were denatured for a 10 minutes, Sammy S.
7/05 protein gel, Kevin and Sammy
Gel 2: induced immediately
Lane 1: Ladder
Lane 2: Strep No IPTG
Lane 3: Strep denatured after 30 min
Lane 4: Strep denatured after 60 min
Lane 5: Strep denatured after 90 min
Lane 6: Strep denatured after 120 min
Lane 7: His No IPTG
Lane 8: His denatured after 30 min
Lane 9: His denatured after 60 min
Lane 10: His denatured after 90 min
Gel 1
Lane 1: Ladder
Lane 2: His 0 min induction
Lane 3: His 30 min induction
Lane 4: His 60 min induction
Lane 5: His 90 min induction
Lane 6: His 120 min induction
Lane 7: Strep 0 min induction
Lane 8: Strep 30 min induction
Lane 9: Strep 60 min induction
Lane 10: Strep 90 min induction
Lane 11: Strep 120 min induction
Lane 12: His 0 min induction 120 min denature
7/09 Egel, Alex
Gel 2: induced immediately
Lane 1: Ladder
Lane 2: Undigested FecR (-) R1A
Lane 3: Pst1 Digested Fec R (-) R1A
Lane 4: Undigested FecR (-) R5B
Lane 5: Pst1 Digested Fec R (-) R5B
Lane 6: Pst1 Digested Fec R (+)
Lane 7: Undigested FecA (-) A1A
Lane 8: Pst1 Digested Fec A (-) A1A
Lane 9: Undigested FecR (-) A5B
Lane 10: Pst1 Digested Fec R (-) A5B
Lane 11: Pst1 Digested Fec R (+) A1B
Lane 12: Pst1 Digested Fec A (+)
7/26/07 Egel, Kevin and Mike
Gel: AIDA after PCR
Lane 1: Ladder
Lane 2: AIDA1+his
Lane 3: AIDA1+strep
Result: We did not get ideal results, though we did get light expression where we expected.
