Team:Harvard/Template:NotebookDataAugust

From 2011.igem.org

(Difference between revisions)
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*Gel stab went into 20µL H2O; 66 C annealing, 3 min elongation; pSB4K5_F+ZFBhl and pSB4K5_R+URA3hl primers
*Gel stab went into 20µL H2O; 66 C annealing, 3 min elongation; pSB4K5_F+ZFBhl and pSB4K5_R+URA3hl primers
*Results: unsuccessful. Perhaps the gel stab did not give an adequate amount of template.
*Results: unsuccessful. Perhaps the gel stab did not give an adequate amount of template.
-
[[File: 2011.08.01. pSB4K5 grad stab(labeled).png|thumb|left|7/29 pSB4K5 gradient samples for gel stab]]
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[[File: HARV2011.08.01. pSB4K5 grad stab(labeled).png|thumb|left|7/29 pSB4K5 gradient samples for gel stab]]
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[[File: 2011.08.01.pSB4K5stab_zeorpoZ_ZF(labeled).png|thumb|none|pSB4K5 gel stab PCR results 8/1/11]]
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[[File: HARV2011.08.01.pSB4K5stab_zeorpoZ_ZF(labeled).png|thumb|none|pSB4K5 gel stab PCR results 8/1/11]]
2) Reverse Touchdown PCR:
2) Reverse Touchdown PCR:
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*3 min elongation
*3 min elongation
*Results: one of the four reactions worked. This is very strange since the reactions should have been identical.
*Results: one of the four reactions worked. This is very strange since the reactions should have been identical.
-
[[File: 2011.08.01.pSB4K5 reverse touchdown(labeled).png|thumb|none|pSB4K5 reverse touchdown 8/1/11]]
+
[[File: HARV2011.08.01.pSB4K5 reverse touchdown(labeled).png|thumb|none|pSB4K5 reverse touchdown 8/1/11]]
3) Template concentration gradient PCR:
3) Template concentration gradient PCR:
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*66 C anneal, 3 min elongation
*66 C anneal, 3 min elongation
*Results: only one of the reactions worked. We did use the gradient cycler which has not always worked well for us. We also noticed that when we removed the tubes from the cycler, most of the liquid had condensed on the lids.
*Results: only one of the reactions worked. We did use the gradient cycler which has not always worked well for us. We also noticed that when we removed the tubes from the cycler, most of the liquid had condensed on the lids.
-
[[File: 2011.08.01.pSB4K5 template conc grad(labeled).png|thumb|none|pSB4K5 template concentration gradient PCR 8/1/11]]
+
[[File: HARV2011.08.01.pSB4K5 template conc grad(labeled).png|thumb|none|pSB4K5 template concentration gradient PCR 8/1/11]]
4) Gradient PCR (overnight):
4) Gradient PCR (overnight):
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*because of a substantial difference in primer melting temperatures, we did a 57-61 C annealing gradient; 90 sec elongation
*because of a substantial difference in primer melting temperatures, we did a 57-61 C annealing gradient; 90 sec elongation
*Results: all 8 conditions worked!
*Results: all 8 conditions worked!
-
[[File: 2011.08.01.ZFBhisura(labeled).png|thumb|none|ZFB-wp-his3-ura3 gradient PCR 8/1/11]]
+
[[File: HARV2011.08.01.ZFBhisura(labeled).png|thumb|none|ZFB-wp-his3-ura3 gradient PCR 8/1/11]]
*Minelute PCR purification of 4 of the reactions: yield=epic.
*Minelute PCR purification of 4 of the reactions: yield=epic.
-
[[File: epic iGEM screen capture.jpeg|thumb|none|BEST YIELD EVER!]]
+
[[File: HARVepic iGEM screen capture.jpeg|thumb|none|BEST YIELD EVER!]]
'''pSR01 plate reading:'''
'''pSR01 plate reading:'''
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{|
{|
-
  |[[File:2011.8.1_cjun_restriction_digest_annotated.png|thumb|We can clearly see the appropriate sized bands (717 and 578 bps) after cutting our cross junction product with Bsa1]]
+
  |[[File:HARV2011.8.1_cjun_restriction_digest_annotated.png|thumb|We can clearly see the appropriate sized bands (717 and 578 bps) after cutting our cross junction product with Bsa1]]
|}
|}
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{|
{|
-
  |[[File:2011.8.1_pos_con_77_f2f3_controls_annotated.png|thumb|All our isothermal assemblies worked, since we can see bands at ~1.4 kb.]]
+
  |[[File:HARV2011.8.1_pos_con_77_f2f3_controls_annotated.png|thumb|All our isothermal assemblies worked, since we can see bands at ~1.4 kb.]]
|}
|}
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{|
{|
-
|[[File:2011.8.1_cb_bottom_plamid_digestion_annotated.png|thumb|Product after digesting our plasmid with Bsa1]]
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|[[File:HARV2011.8.1_cb_bottom_plamid_digestion_annotated.png|thumb|Product after digesting our plasmid with Bsa1]]
|}
|}
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**Performed PCR and ran gel on products
**Performed PCR and ran gel on products
***Successful!!
***Successful!!
-
[[File: 2011.08.01.Zeocincheckfroplatereader(labeled).png|thumb|none|8/1/11 Zeocin insertion into ECNR2 for plate reader]]
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[[File: HARV2011.08.01.Zeocincheckfroplatereader(labeled).png|thumb|none|8/1/11 Zeocin insertion into ECNR2 for plate reader]]
====Transformation of Zif268 into ECNR2+zeocin without Kan+ZFB+wp====
====Transformation of Zif268 into ECNR2+zeocin without Kan+ZFB+wp====
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***This served as a postive control
***This served as a postive control
***Grown in .005% SDS and 2,4,8,12,16 µM of IPTG
***Grown in .005% SDS and 2,4,8,12,16 µM of IPTG
-
*Plate was taken down to plate reader at 5:25 pm and began a 16 hour program
+
*Plate was taken down to plate reader at 5:25 pm and began a 16 hour program</div>
-
 
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<div id='802' style="display:none">
==August 2nd==
==August 2nd==
===Team TolC===
===Team TolC===
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{|
{|
-
|[[File:2011.8.2_miniprep_comparison.jpg|thumb|Miniprep results. PZE22G had excellent yields, while CB Bottom did not.]]
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|[[File:HARV2011.8.2_miniprep_comparison.jpg|thumb|Miniprep results. PZE22G had excellent yields, while CB Bottom did not.]]
|}
|}
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{|
{|
-
|[[File:2011.8.2_oligo_digestion_1_annotated.png|thumb|Our first digestion did not show any bands below 100 bp.]]
+
|[[File:HARV2011.8.2_oligo_digestion_1_annotated.png|thumb|Our first digestion did not show any bands below 100 bp.]]
|}
|}
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{|
{|
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|[[File:2011.8.2_oligo_digestion_2_annotated.png|thumb|The bands on this 1.2% e-gel were unexpectedly high. It maybe a problem with the gel.]]
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|[[File:HARV2011.8.2_oligo_digestion_2_annotated.png|thumb|The bands on this 1.2% e-gel were unexpectedly high. It maybe a problem with the gel.]]
|}
|}
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*We got some beautiful growth curves! A few of the more critical ones are below:
*We got some beautiful growth curves! A few of the more critical ones are below:
*NM+his: both selection strain and pSR01 grew, but the selection strain grew faster. This might just be an artifact from having accidentally added more cells to the selection strain than the pSR01 wells, but it may also show that the pSR01 strain grows slower. Another possibility is that we are expressing enough zinc fingers for it to be slightly toxic for the cells. Next time maybe we should let the plate reader go for longer than 16 hours just in case the cells need more time.
*NM+his: both selection strain and pSR01 grew, but the selection strain grew faster. This might just be an artifact from having accidentally added more cells to the selection strain than the pSR01 wells, but it may also show that the pSR01 strain grows slower. Another possibility is that we are expressing enough zinc fingers for it to be slightly toxic for the cells. Next time maybe we should let the plate reader go for longer than 16 hours just in case the cells need more time.
-
[[File: 2011.08.01.NM+his.png|none|NM+his: 162 and 172 selection strain, 182 and 192 pSR01]]
+
[[File: HARV2011.08.01.NM+his.png|none|NM+his: 162 and 172 selection strain, 182 and 192 pSR01]]
*NM: selection strain does not grow, while pSR01 does a perfect rescue!
*NM: selection strain does not grow, while pSR01 does a perfect rescue!
[[File: 2011.08.01.NM.png|none|NM: 163 and 173 selection strain, 183 and 193 pSR01]]
[[File: 2011.08.01.NM.png|none|NM: 163 and 173 selection strain, 183 and 193 pSR01]]
-
*pSR01 3-AT comparison: the concentration of 3-AT does not seem to make a difference in the growth curves (the differences appear to be due to different wells starting with slightly different amounts of cells). Next time we can try a higher 3-AT concentration, but since we expect Zif268 to bind strongly this might just be what you get.
+
*pSR01 HARV3-AT comparison: the concentration of 3-AT does not seem to make a difference in the growth curves (the differences appear to be due to different wells starting with slightly different amounts of cells). Next time we can try a higher 3-AT concentration, but since we expect Zif268 to bind strongly this might just be what you get.
-
[[File: 2011.08.01.pSR01 comparison.png|none|pSR01 colony comparison: 182 NM+his, 183 NM, 184 1mM 3-AT, 185 2.5mM 3-AT, 186 5mM 3-AT]]
+
[[File: HARV2011.08.01.pSR01 comparison.png|none|pSR01 colony comparison: 182 NM+his, 183 NM, 184 1mM 3-AT, 185 2.5mM 3-AT, 186 5mM 3-AT]]
*NM+his+IPTG: IPTG more or less killed the pSR01 cells but not the selection strain. This may be caused by too many zinc fingers being expressed, so we may want to try less IPTG.
*NM+his+IPTG: IPTG more or less killed the pSR01 cells but not the selection strain. This may be caused by too many zinc fingers being expressed, so we may want to try less IPTG.
-
[[File: 2011.08.01.NM+his+IPTG.png|none|NM+his+IPTG: 167 and 177 selection strain, 187 and 197 pSR01]]
+
[[File: HARV2011.08.01.NM+his+IPTG.png|none|NM+his+IPTG: 167 and 177 selection strain, 187 and 197 pSR01]]
'''EcNR2 Wolfe Selection system: MAGE'''
'''EcNR2 Wolfe Selection system: MAGE'''
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'''pSB4K5:'''
'''pSB4K5:'''
*Our pSB4K5 PCR still eludes us. Our overnight gradient did work, but no better than the other days' reactions.
*Our pSB4K5 PCR still eludes us. Our overnight gradient did work, but no better than the other days' reactions.
-
[[File: 2011.08.02.pSB4K5 gradient pcr1(labeled).png|thumb|none|pSB4K5 gradient 8/2/11]]
+
[[File: HARV2011.08.02.pSB4K5 gradient pcr1(labeled).png|thumb|none|pSB4K5 gradient 8/2/11]]
-
*Gel extraction and Minelute only gave us a 9.4ng/µL yield (260/280=1.82) which is not enough for isothermal assembly. We will have to miniprep more plasmid for template for PCRs, and we might want to consider using ligation or some other method.
+
*Gel extraction and Minelute only gave us a 9.4ng/µL yield (260/280=1.82) which is not enough for isothermal assembly. We will have to miniprep more plasmid for template for PCRs, and we might want to consider using ligation or some other method.</div>
-
 
+
<div id='803' style="display:none">
==August 3rd==
==August 3rd==
===Team ZF===
===Team ZF===
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We ran a gel of our digestion product from yesterday, and our PCR to clone out Zif 268 to construct the reporter plasmid.
We ran a gel of our digestion product from yesterday, and our PCR to clone out Zif 268 to construct the reporter plasmid.
{|
{|
-
|[[File:2011.8.8_digest_PCR_annotated.png|thumb|Our results for the final digestion we ran yesterday, and the PCR to clone out Zif 268.]]
+
|[[File:HARV2011.8.8_digest_PCR_annotated.png|thumb|Our results for the final digestion we ran yesterday, and the PCR to clone out Zif 268.]]
|}
|}
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{|
{|
-
|[[File:2011.8.3_pc77_swap_minipreps.jpg|thumb|Results of our miniprep.]]
+
|[[File:HARV2011.8.3_pc77_swap_minipreps.jpg|thumb|Results of our miniprep.]]
|}
|}
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{|
{|
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|[[File:2011.8.3_junction_pcr_annotated.png|thumb]]
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|[[File:HARV2011.8.3_junction_pcr_annotated.png|thumb]]
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|[[File:2011.8.3_junction_pcr_2_and_gfp_annotated.png|thumb]]
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|[[File:HARV2011.8.3_junction_pcr_2_and_gfp_annotated.png|thumb]]
|}
|}
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{|
{|
-
|[[File:2011.8.3_junction_pcr_2_and_gfp_annotated.png|thumb|The last four lanes show the expected band size of ~825 bp for GFP. However the bands are comparatively faint.]]
+
|[[File:HARV2011.8.3_junction_pcr_2_and_gfp_annotated.png|thumb|The last four lanes show the expected band size of ~825 bp for GFP. However the bands are comparatively faint.]]
|}
|}
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{|
{|
-
|[[File:2011.8.3_zif_clone_and_gfp_annotated.png|thumb|We made a 1% agarose gel to gel purify our zif clone and GFP. The bands for GFP are much fainter than those for Zif 268.]]
+
|[[File:HARV2011.8.3_zif_clone_and_gfp_annotated.png|thumb|We made a 1% agarose gel to gel purify our zif clone and GFP. The bands for GFP are much fainter than those for Zif 268.]]
|}
|}
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{|
{|
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|[[File:2011.8.3_gfp_for_gel_extr._annotated.png|thumb|We ran another PCR to get out GFP.]]
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|[[File:HARV2011.8.3_gfp_for_gel_extr._annotated.png|thumb|We ran another PCR to get out GFP.]]
|}
|}
   
   
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{|
{|
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|[[File:2011.8.3_double_digested_annotated.png|thumb|We could see bands of the appropriate size, after digesting our PCR product. Unpure = cut without PCR purification.]]
+
|[[File:HARV2011.8.3_double_digested_annotated.png|thumb|We could see bands of the appropriate size, after digesting our PCR product. Unpure = cut without PCR purification.]]
|}
|}
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**PCR failed
**PCR failed
-
[[File:2011.08.03.pSB4K5 rtd.png|thumb|none|A successful PCR would have resulted in a 3.5-4Kb band.]]
+
[[File:HARV2011.08.03.pSB4K5 rtd.png|thumb|none|A successful PCR would have resulted in a 3.5-4Kb band.]]
*Miniprep of pSB4K5 strain for future PCR yielded 15ng/µL
*Miniprep of pSB4K5 strain for future PCR yielded 15ng/µL
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===Team TolC===
===Team TolC===
-
*Set up and ran plate reader for the growth rates of ECNR2+kan-ZFB-wp+zeocin+spec plasmid, ECNR2+kan-ZFB-wp+zeocin Δspec plasmid, ECNR2, and ECNR2+zeocin+spec plasmid Δkan-ZFB-wp in .04% SDS and 25, 12.5, 6.25, 3.125, 1.5625 µM
+
*Set up and ran plate reader for the growth rates of ECNR2+kan-ZFB-wp+zeocin+spec plasmid, ECNR2+kan-ZFB-wp+zeocin Δspec plasmid, ECNR2, and ECNR2+zeocin+spec plasmid Δkan-ZFB-wp in .04% SDS and 25, 12.5, 6.25, 3.125, 1.5625 µM</div>
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<div id='804' style="display:none">
==August 4th==
==August 4th==
===Team ZF===
===Team ZF===
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{|
{|
-
|[[File:2011.8.4_xba1_digest_and_ligation_annotated.png|thumb|Comparing our original, cut, and ligated cross junction.]]
+
|[[File:HARV2011.8.4_xba1_digest_and_ligation_annotated.png|thumb|Comparing our original, cut, and ligated cross junction.]]
|}
|}
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{|
{|
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|[[File:2011.8.4_cb_bottom_for_extraction_annotated.png|thumb|A gel of the CB Bottom backbone, after digestion. We expected a band at ~2.7 kb]]
+
|[[File:HARV2011.8.4_cb_bottom_for_extraction_annotated.png|thumb|A gel of the CB Bottom backbone, after digestion. We expected a band at ~2.7 kb]]
|}
|}
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*Looks great!
*Looks great!
*1mM, 2.5mM, and 5mM 3-AT have slight differences in growth rate, but it is hard to tell how significant they are. However, starting with 10mM 3-AT, the growth rate drops dramatically! Here is an example from 1 culture set:
*1mM, 2.5mM, and 5mM 3-AT have slight differences in growth rate, but it is hard to tell how significant they are. However, starting with 10mM 3-AT, the growth rate drops dramatically! Here is an example from 1 culture set:
-
[[File: 2011.08.03.pSR01 1(2) 3-AT gradient.png|none|pSR01 culture 1 (duplicate 2) 3-AT gradient]]
+
[[File: HARV2011.08.03.pSR01 1(2) 3-AT gradient.png|none|pSR01 culture 1 (duplicate 2) 3-AT gradient]]
'''MAGE 3 HisB and PyrF Knock Out Gel'''
'''MAGE 3 HisB and PyrF Knock Out Gel'''
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**Similar to HisB--all the lanes had bands but some were brighter than others.
**Similar to HisB--all the lanes had bands but some were brighter than others.
-
[[File:2011.08.04 HisBUra3 MAGE 3 Deletion Check (labeled).png|thumb|none|HisB]]
+
[[File:HARV2011.08.04 HisBUra3 MAGE 3 Deletion Check (labeled).png|thumb|none|HisB]]
-
[[File:2011.08.04 PyrF MAGE 3 Deletion Check 2s exposure (labeled).png|thumb|none|PyrF]]
+
[[File:HARV2011.08.04 PyrF MAGE 3 Deletion Check 2s exposure (labeled).png|thumb|none|PyrF]]
'''ZFB-wp-his3-ura3 restriction enzyme PCR'''
'''ZFB-wp-his3-ura3 restriction enzyme PCR'''
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====Plate Reader====
====Plate Reader====
{|
{|
-
|[[File:2011.8.4graphofKNplatereader.png|thumb|Plate Reader Graph of KN.]]
+
|[[File:HARV2011.8.4graphofKNplatereader.png|thumb|Plate Reader Graph of KN.]]
|}
|}
{|
{|
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|[[File:2011.8.4graphofWNplatereader.png|thumb|Plate Reader Graph of WN.]]
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|[[File:HARV2011.8.4graphofWNplatereader.png|thumb|Plate Reader Graph of WN.]]
|}
|}
{|
{|
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|[[File:2011.8.4graphofKSplatereader.png|thumb|Plate Reader Graph of KS.]]
+
|[[File:HARV2011.8.4graphofKSplatereader.png|thumb|Plate Reader Graph of KS.]]
|}
|}
{|
{|
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|[[File:2011.8.4graphofWSplatereader.png|thumb|Plate Reader Graph of WS.]]
+
|[[File:HARV2011.8.4graphofWSplatereader.png|thumb|Plate Reader Graph of WS.]]
|}
|}
====PCR of plate reader colonies====
====PCR of plate reader colonies====
*Made glycerol stock of ECNR2+spec plasmid+zeocin, WS</div>
*Made glycerol stock of ECNR2+spec plasmid+zeocin, WS</div>

Revision as of 22:05, 19 September 2011