Team:Harvard/Template:NotebookDataJuly4

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(Transformation plating results)
 
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===Team ZF===
===Team ZF===
====Result of OZ052 and OZ123 Transformation====
====Result of OZ052 and OZ123 Transformation====
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[[#Transformation for OZ plasmids|Last Friday]] we plated out the transformed bacteria for the two OZ controls in 10 ul and 50 ul volumes.  They both yielded colonies, but the 10 ul plates only had a handful while the 50 ul plates had around 15-20 colonies each.  We picked four colonies from each plate and grew them in LB/spec so that tomorrow we can perform a miniprep and send them off for sequencing.  We should know the final verdict by Wednesday regarding which ones are good or bad.
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Last Friday we plated out the transformed bacteria for the two OZ controls in 10 ul and 50 ul volumes.  They both yielded colonies, but the 10 ul plates only had a handful while the 50 ul plates had around 15-20 colonies each.  We picked four colonies from each plate and grew them in LB/spec so that tomorrow we can perform a miniprep and send them off for sequencing.  We should know the final verdict by Wednesday regarding which ones are good or bad.
====Glycerol stockmaking====
====Glycerol stockmaking====
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We got the sequencing results back from last Friday, and took the following colonies to make into glycerol stock solutions: 77.1, 79.2, 80.1, 81.1 (x2 for both FH bottom and Myc 198), 82.1, 83.4, 84.1, Zif268.  These colonies are a subset of the "good" colonies from our [[Lab_Notebook:_July#Sequencing_Results|sequencing results]] from Sunday.  We mixed 120 ul of bacteria in mid-log with 300 ul of 80% glycerol to make the final solution, which was stored at -80 degrees C.
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We got the sequencing results back from last Friday, and took the following colonies to make into glycerol stock solutions: 77.1, 79.2, 80.1, 81.1 (x2 for both FH bottom and Myc 198), 82.1, 83.4, 84.1, Zif268.  These colonies are a subset of the "good" colonies from our sequencing results from Sunday.  We mixed 120 ul of bacteria in mid-log with 300 ul of 80% glycerol to make the final solution, which was stored at -80 degrees C.
====Restriction enzyme test====
====Restriction enzyme test====
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{|
{|
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  |[[File:2011.7.25.enzyme_test_1.png|thumb|E-gel of our first digestion. The gel looks unusual (notice the bubble-like formation), so we performed a second digestion.]]
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  |[[File:HARV2011.7.25.enzyme_test_1.png|thumb|E-gel of our first digestion. The gel looks unusual (notice the bubble-like formation), so we performed a second digestion.]]
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  |[[File:2011.7.25_enzyme_test_take2.png|thumb|1% agarose gel of our second digestion. Although the bands are faint, we can see the appropriately sized bands for Bsa1.]]
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  |[[File:HARV2011.7.25_enzyme_test_take2.png|thumb|1% agarose gel of our second digestion. Although the bands are faint, we can see the appropriately sized bands for Bsa1.]]
|}
|}
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*OZ123 - 76, 126</div>
*OZ123 - 76, 126</div>
<div id="726" style="display:none">
<div id="726" style="display:none">
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==July 26==
==July 26==
===Team ZF===
===Team ZF===
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{|
{|
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|[[File:2011.7.26_ultramer_reaction_and_restriction_rerun_annotated.png|thumb|Our first PCR was extremely streaky, so we decided to redo it.]]
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|[[File:HARV2011.7.26_ultramer_reaction_and_restriction_rerun_annotated.png|thumb|Our first PCR was extremely streaky, so we decided to redo it.]]
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|[[File:2011.7.26_ultramer_touchdowns_last_four_lanes_annotated.png|thumb|We did a touchdown PCR, and our product looks better, although it's still streaky.]]
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|[[File:HARV2011.7.26_ultramer_touchdowns_last_four_lanes_annotated.png|thumb|We did a touchdown PCR, and our product looks better, although it's still streaky.]]
|}
|}
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{|
{|
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|[[File:2011.7.26_PCR_purification_after_ultramer_assembly.jpg|thumb|Concentrations of our PCR product after PCR purification. This was used in our isothermal assembly.]]
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|[[File:HARV2011.7.26_PCR_purification_after_ultramer_assembly.jpg|thumb|Concentrations of our PCR product after PCR purification. This was used in our isothermal assembly.]]
|}
|}
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{|
{|
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|[[File:2011.7.26_Miniprep.jpg|thumb|Our OZ concentrations after miniprep.]]
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|[[File:HARV2011.7.26_Miniprep.jpg|thumb|Our OZ concentrations after miniprep.]]
|}
|}
The miniprep lysis step looked good, and the samples turned blue after lysis.
The miniprep lysis step looked good, and the samples turned blue after lysis.
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We then performed a junction PCR using the [[Lab_Notebook:_July#PCR_of_expression_plasmid_cross-junction|previous protocol]], and checked the products on a gel:
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We then performed a junction PCR using the previous protocol, and checked the products on a gel:
{|
{|
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|[[File:2011.7.26_oz052_and_oz123_for_seq_annotated.png|thumb|Gel of our PCR product that we sent out for sequencing.]]
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|[[File:HARV2011.7.26_oz052_and_oz123_for_seq_annotated.png|thumb|Gel of our PCR product that we sent out for sequencing.]]
|}
|}
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*Each template was analysed with presence of different sized bands for rpoZ (using rpoZ_F and rpoZ_R) and presence of Zeocin cassette (using rpoZ_R and zeocin_R)
*Each template was analysed with presence of different sized bands for rpoZ (using rpoZ_F and rpoZ_R) and presence of Zeocin cassette (using rpoZ_R and zeocin_R)
*Gel resulted with the following:
*Gel resulted with the following:
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[[File: 2011.07.26.Zeocininsertion(labeled).png|thumb|none|Zeocin insert check 7/26/11]]
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[[File: HARV2011.07.26.Zeocininsertion(labeled).png|thumb|none|Zeocin insert check 7/26/11]]
**'''SUCCESS!!! Yippee!!!'''
**'''SUCCESS!!! Yippee!!!'''
**NOTE. Since there was a bit of non specific annealing, we might want to use a higher annealing temperature of 64C instead of 62C
**NOTE. Since there was a bit of non specific annealing, we might want to use a higher annealing temperature of 64C instead of 62C
*Decided to run PCR for the Kan insertion just to make sure it was still present in the 4 colonies we chose, and it is!!!
*Decided to run PCR for the Kan insertion just to make sure it was still present in the 4 colonies we chose, and it is!!!
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[[File: 2011.07.26.kaninsertioncheck(labeled).png|thumb|none|Kan-ZFB-wp insert check 7/26/11]]
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[[File: HARV2011.07.26.kaninsertioncheck(labeled).png|thumb|none|Kan-ZFB-wp insert check 7/26/11]]
WERE READY TO ROOOCCKKK!!!!!
WERE READY TO ROOOCCKKK!!!!!
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{|
{|
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  |[[File:2011.7.28_practice_plate_primer_test_annotated.png|thumb|Practice plate primer test]]
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  |[[File:HARV2011.7.28_practice_plate_primer_test_annotated.png|thumb|Practice plate primer test]]
  |}
  |}
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*PCR: colonies/culture diluted 1:20 as template (and colonies grown up simultaneously); M13_F and M13_R primers; 50˚C annealing and 3min elongation; 25 cycles
*PCR: colonies/culture diluted 1:20 as template (and colonies grown up simultaneously); M13_F and M13_R primers; 50˚C annealing and 3min elongation; 25 cycles
*Results: bands are similar to the ones seen the last time we did this PCR (and those samples had also been sequence-verified). These colonies should be good to go!
*Results: bands are similar to the ones seen the last time we did this PCR (and those samples had also been sequence-verified). These colonies should be good to go!
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[[File: 2011.07.29.pSR01check(labeled).png|thumb|none|pSR01 check 7/29/11]]
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[[File: HARV2011.07.29.pSR01check(labeled).png|thumb|none|pSR01 check 7/29/11]]
'''pSB4K5:'''
'''pSB4K5:'''
*Miniprepped both cultures. Yields weren't great (in the case of 2:17H, there actually was a spill) but they are still usable.
*Miniprepped both cultures. Yields weren't great (in the case of 2:17H, there actually was a spill) but they are still usable.
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**58˚C anneal, 3 min elongation, 25 cycles
**58˚C anneal, 3 min elongation, 25 cycles
*Results: the product was really really really faint--primer dimers were by far the bulk of the product.  We realized this is probably because one of the primers includes a Not1 restriction site, which is palindromic, 8 bases long, and all Cs and Gs.
*Results: the product was really really really faint--primer dimers were by far the bulk of the product.  We realized this is probably because one of the primers includes a Not1 restriction site, which is palindromic, 8 bases long, and all Cs and Gs.
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[[File: 2011.07.29.pSB4K5(labeled).png|thumb|none|pSB4K5 PCR to add homology 7/29/11]]
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[[File: HARV2011.07.29.pSB4K5(labeled).png|thumb|none|pSB4K5 PCR to add homology 7/29/11]]
*In order to counteract the primer dimers, we will try a gradient PCR with a range of higher annealing temperatures:
*In order to counteract the primer dimers, we will try a gradient PCR with a range of higher annealing temperatures:
**same protocol as before except the annealing temp went from 58-65 with 8 samples (1=58, 8=65)
**same protocol as before except the annealing temp went from 58-65 with 8 samples (1=58, 8=65)
**Results: 3:11P did have visible product bands, but the primer dimers are still stronger
**Results: 3:11P did have visible product bands, but the primer dimers are still stronger
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[[File: 2011.07.29 pSB4K5 grad(labeled).png|thumb|none|pSB4K5 gradient PCR 7/29/11]]
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[[File: HARV2011.07.29 pSB4K5 grad(labeled).png|thumb|none|pSB4K5 gradient PCR 7/29/11]]
===Team TolC===
===Team TolC===
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*So we chose 5 colonies and ran PCR on them to gel them out
*So we chose 5 colonies and ran PCR on them to gel them out
**The gel shows success for colonies 2,3,4, and 5 that we chose!!!
**The gel shows success for colonies 2,3,4, and 5 that we chose!!!
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[[File: 2011.07.29.zif268plasmidcheck1.png|thumb|none|Zif268 plasmid insertion check 7/29/11]]
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[[File: HARV2011.07.29.zif268plasmidcheck1.png|thumb|none|Zif268 plasmid insertion check 7/29/11]]
'''READY FOR SELECTION!!!'''
'''READY FOR SELECTION!!!'''
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===Team ZF===
===Team ZF===
====Transformation plating results====
====Transformation plating results====
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We got no growth on any of the plated transformed bacteria for either the ElectroComp or ChemComp bacteria.  To check whether the isothermal assembly itself was the problem, we ran a junction PCR using our [[Lab_Notebook:_July#PCR_of_expression_plasmid_cross-junction|previous protocol]] on the isothermal assembly mix, along with a positive control (Myc981):
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We got no growth on any of the plated transformed bacteria for either the ElectroComp or ChemComp bacteria.  To check whether the isothermal assembly itself was the problem, we ran a junction PCR using our previous protocol on the isothermal assembly mix, along with a positive control (Myc981):
{|
{|
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  |[[File:2011.7.29_isothermal_assembly_junction_test_annotated.png|thumb|No bands visible for the isothermal assembly]]
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  |[[File:HARV2011.7.29_isothermal_assembly_junction_test_annotated.png|thumb|No bands visible for the isothermal assembly]]
  |}
  |}
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{|
{|
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  |[[File:2011.7.29_Practice_Plate_oligo_pcr_pure.jpg|thumb|DNA concentrations]]
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  |[[File:HARV2011.7.29_Practice_Plate_oligo_pcr_pure.jpg|thumb|DNA concentrations]]
  |}
  |}
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{|
{|
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  |[[File:2011.7.29_practice_plate_neg_controls_annotated.png|thumb|Strange results]]
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  |[[File:HARV2011.7.29_practice_plate_neg_controls_annotated.png|thumb|Strange results]]
  |}
  |}
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{|
{|
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  |[[File:2011.7.30_CB_bottom_miniprep.jpg|thumb|DNA concentrations after miniprep]]
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  |[[File:HARV2011.7.30_CB_bottom_miniprep.jpg|thumb|DNA concentrations after miniprep]]
  |}
  |}
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{|
{|
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  |[[File:2011.7.31_primer_tag_test_two_annotated.png|thumb|PCR repeat results]]
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  |[[File:HARV2011.7.31_primer_tag_test_two_annotated.png|thumb|PCR repeat results]]
  |}
  |}

Latest revision as of 19:22, 3 August 2011