Difference between revisions of "Team:Warwick/Project5"

 
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{{WarwickTimeline}}
 
{{WarwickTimeline}}
 
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<div class="cd-timeline-content">
 
<div class="cd-timeline-content">
 
<h2>Week 2</h2>
 
<h2>Week 2</h2>
−
<p>7/7/15
+
<p>
−
 
+
−
<br>We made 8 plates of streptomycin and chloramphenicol  ­ 3 plates of chloramphenicol ­ Cells made electrocompetent following lab protocol
+
−
 
+
−
<br>8/7/15
+
−
 
+
−
­ <br>Cells grew!
+
−
<br> <b>Cells transformed-</b> ­
+
−
 
+
  
−
<br>INP 8H psB1c3 5
+
<br>07/07/2015:
  
−
<br>Ipp OmpA 6L psB1c4 5
+
- <br>Agar plates (with chloramphenicol and streptomycin) made.
  
−
<br>Zif 23 1G psB1c5 3
+
- <br>Top 10 cells made electrocompetent.
  
−
<br>9/7/15  ­ <br>Met Miriam ­ phD presentation and explanation of cloning/ gibson assembly/pcr
+
<br>08/07/2015:
  
−
­ <br>4 minipreps  ­ Nanodrop results: J04450 ­ 55.1ng/l
+
- <br>Transformed Top 10 cells with INP, Lpp OmpA and Zif 23 from 2015 DNA
  
−
                              <br>INP ­ 10.8 ng/l
+
Distribution Kit.
 +
<p><img src="https://static.igem.org/mediawiki/2015/0/01/Distribution_Kit_table_Lab_Book.png" height="60px" width="400px" border="1px"></p>
 +
<br>09/07/2015:
  
−
                            <br> C2001 ­ 16.1 ng/l
+
- <br>Mini-prepped cells from yesterday.
  
−
                            <br> LPPOMPA ­ 38.4 ng/l ­ Electrophoresis: Made 10% urea, 1% agarose gel ethidum gel
+
- <br>Nanodrop results for mini-prepped plasmids.
 +
<p><img src="https://static.igem.org/mediawiki/2015/7/7b/NanoDropResultsTableLab_BookiGEMWarwick.png" height="60px" width="400px" border="1px"></p>
 +
<br>Ran an electrophoresis gel with plasmids from the mini-prep.
  
−
                          <br>Put plasmids from mini prep in gel, ran for 30 mins at 100V
 
  
−
­ (Left to right: Ladder, LPPOMPA, C2001, INP, J04450)
 
 
</p>
 
</p>
 
 
Line 99: Line 89:
 
<p>
 
<p>
  
−
<br>13/7/15
 
  
−
­ <br>Grow up MG1655 (Z1) cells. ­ Put into 3 ml LB
+
­ <br>13/07/2015:
  
−
­ <br>Added 3l Strep
+
- <br>Grew up MG1655-Z1 cells (added Streptomycin).
  
−
­ <br>Put into shaking incubator (37oC) overnight.
+
<br>14/07/2015:
 +
 
 +
- <br>Made MG1655-Z1 cells competent.
  
−
<br>14/7/15
 
  
−
­ <br>Made competent cells (followed competent cells protocol)
 
  
 
</p>
 
</p>
Line 129: Line 118:
  
  
 +
<br>22/07/2015:
  
−
<br>22/7/15
+
- <br>Ligated full construct gBlock into pSB1C3 plasmid backbone.
  
−
­ <br>Ligated full construct g­block into Psb1C3 plasmid backbone.
+
<br>23/07/2015:
  
−
<br>23/7/15
+
- <br>Transformed ligated plasmid into Top 10 cells.
  
−
­ <br>Diluted primers to 100mM, made set of 5mM primer tubes. ­ Transformed ligated plasmid into top 10 cells (still some plasmid left in the top shelf of
+
- <br>Ran PCR of gBlocks.
  
−
the freezer). ­ PCR: 5l 5mM forward primer
+
<br>24/07/2015:
  
−
        <br> 5l 5mM reverse primer
+
- <br>Transformed ligated plasmid into Top 10 cells again.
  
−
        <br> 0.5l of g­block
+
- <br>Ran PCR of BclA, INP and pgsA gBlocks.
  
−
      <br>  25l of Q5 mastermix
+
- <br>Transformed more cells with ligated plasmid (using electroporation).
−
 
+
−
        <br> 14.5l of water
+
−
 
+
−
<br>24/7/15
+
−
 
+
−
<br>­ Ligated plasmid transformed cells did not grow overnight, repeating today. ­ <br>PCRing the bcla, inp and pgsa. ­<br> Transformed more cells with ligated plasmid using electroporation, plated. ­<br> Also PCRd zf10, zf14 and zf2.
+
  
 +
- <br>Plated transformed cells.
  
 +
- <br>Ran PCR of sZF2, sZF10 and sZF14.
  
  
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<div class="cd-timeline-content">
 
<div class="cd-timeline-content">
 
<h2>Week 5</h2>
 
<h2>Week 5</h2>
−
<p>Both of the chemically transformed plates and electrotransformed plate grew over weekend.
+
<p>
−
<br>Lpp­ompA­zif268: colony PCR of clone 2 ok (inoculate culture to purify DNA ­ inoculate 2
+
 
 +
<br>27/07/2015:
 +
 
 +
- <br>Chemically transformed plates and electrotransformed plated grew over  
 +
 
 +
weekend.
 +
 
 +
<br>28/07/2015
 +
 
 +
- <br>Colony PCR of the 2 clones.
 +
 
 +
<br>29/07/2015
 +
 
 +
- <br>Conducted plasmid mini prep of bacterial cultures.
 +
 
 +
- <br>Ran gel electrophoresis of redone PCR.
 +
 
 +
- <br>Ran gel electrophoresis of mini prep restriction digestion.
 +
 
 +
- <br>Redoing PCR (of INP, sZF2, sZF10 and sZF14) with DMSO (at 0%, 3% and
 +
 
 +
6%).
 +
 
 +
- <br>PCR purification of BclA and  and pgsA.
 +
 
 +
<br>30/07/2015
 +
 
 +
- <br>Re-did PCR of INP, sZF10 and sZF14.
 +
 
 +
- <br>sZF14 returned positive (concentration of 31.8 ng/µl after purification).
 +
 
 +
- <br>Purified sZF2.
 +
 
 +
- <br>Re-doing PCR of INP and sZF10.
  
−
cultures, 4.5ml each. Miniprep them after making a glycerol stock).
+
<br>31/07/2015
−
<br>Conducted plasmid miniprep of bacterial culture. ­ Gel electrophoresis of redone PCR ­ Pgsa worked, zf2 (all others need to be repeated at
+
  
−
a later stage). ­ <br>Running gel of miniprep restriction digestion. ­ Re­doing PCR: INP, zf2, zf14 (67oC). zf10 (69oC) ­ with DMSO (0%, 3%, 6%). ­<br> PCR purification of BCLA and PGSA (nanodrop returned negative, redoing). ­ Redoing PCR of INP, zf2, zf10, zf14.
+
- <br>Gel of sZF10 and INP PCR product returned negative.  
−
<br>Redid PCR of INP, zf10 and zf14. ­ zf14 returned positive, purified (31.8 ng/l. ­ Redoing PCR of INP and zf10. ­ Purified zf2, nanodropped.
+
−
<br>Gel of zf10 and INP returned negative.
+
−
<br>Redoing PCR with 66oC and 680C annealing temperatures.  
+
  
−
<br>Restriction digestion protocol: 5l cutsmart 10x
+
- <br>Re-doing PCR with 66 ̊ C and 68 ̊ C annealing temperatures.  
−
                                                            1l DNA
+
−
                                                            10 units enzyme each
+
−
                                                            make up to 50l water
+
−
<br>For Nde1-Age1-HF, incubate at 370C for 5-15 minutes.
+
−
<br>Diluted INP g-block and ZF10 g-block by a factor of 10.
+
  
−
<br>Ran gradient PCR of INP and ZF10 (made 12 tubes of 20l).
+
- <br>Ran gradient PCR of sZF10 and INP.  
  
 +
- <br>Did restriction digestion of BclA, pgsA and Lpp OmpA
 
</p>
 
</p>
 
 
Line 204: Line 215:
 
<h2>Week 6</h2>
 
<h2>Week 6</h2>
 
<p>
 
<p>
−
<br>3/8/15
+
<br>03/08/2015:
  
−
<br>Redoing restriction digest of LPP-OMPA
+
- <br>Re-did restriction digestion of Lpp OmpA.
−
<br>Running gel from Friday (last time = 6.1 ng/l, issue with plasmid?)
+
−
<br>Plasmid concentration only 5.9ng/l, running ligation anyway.
+
−
<br>20l ligation reaction (full details in book).
+
  
−
<br>4/8/15
+
- <br>Ligated BclA and pgsA into pSB1C3.
  
−
<br>Transforming ligated plasmid into mg1655 cells.
+
<br>04/08/2015:
−
<br>PCR purify INP.
+
−
<br>Restriction digestion of INP and LPP-OMPA.
+
−
<br>Diluted full construct g-block 10x.
+
−
<br>Gradient PCR of LPP-OMPA full construct.
+
  
−
<br>5/5/15
+
- <br>Transformed ligated plasmid into MG1655-Z1 cells.
  
−
<br>2 colonies from a grown DH5 Z1 plate were selected, 2 cultures inoculated, grown in shaking incubator.
+
- <br>PCR purified INP.  
−
<br>Purify and restriction digest full construct.
+
−
<br>INP digestion.
+
−
<br>Streaked plate with PGSA and BCLA transformed cells.
+
  
−
<br>6/8/15
+
- <br>Did a restriction digestion of INP and Lpp OmpA.
  
−
<br>ZF10 gel electrophoresis.
+
- <br>Ran a gradient PCR of Lpp OmpA full construct.
−
<br>Cut out ZF10 (at 270 bases, according to ladder).
+
−
<br>Gel extraction (freeze ‘n’ squeeze).
+
  
−
<br>7/8/15
+
<br>05/08/2015:
  
−
<br>Streaked chlor plates with transformed RFP DH5 Z1 cells.
+
- <br>2 cultures inoculated with colonies from a DH5α Z1 plate.
−
<br>Re-streaked plates with BCLA and PGSA transformed cells.
+
−
<br>Made chemically competent DH5 Z1 cells (frozen at -80oC).
+
−
vRan gel of digested plasmid, only obtained single band at 2000 base pairs (where backbone without RFP would be, lacking band at 1000 base pairs (for RFP gene)).
+
−
Ran gel of undigested plasmids, single band at 3000 base pairs.
+
−
Gel purified digested plasmid, nanodropped (2.1ng/l).
+
  
 +
- <br>Purified and restriction digested Lpp OmpA full construct.
  
−
<br>8/8/15
+
- <br>Restriction digestion of INP.
  
−
<br>RFP and BCLA transformed cells grow with single colonies.
+
- <br>Streaked plate with pgsA and BclA transformed cells.  
−
<br>PGSA plate showed no growth.
+
−
<br>Inoculated falcon tubes with RFP and BCLA. Also inoculated tube using old PGSA, incubating in shaking incubator.
+
−
Plated pure DH5 Z1 on a chlor and a clean plate (to establish background growth).
+
  
−
<br>9/8/15
+
<br>06/08/2015:
  
−
<br>Miniprep of RFP, BCLA and PGSA transformed cells.
+
- <br>Gel electrophoresis of sZF10.
−
<br>Chlor control plate grew no colonies, clean control plate grew a lawn.  
+
  
 +
- <br>Cut out band corresponding to sZF10 (at 270 bases).
 +
 +
- <br>Did a gel extraction (using Freeze ‘n’ Squeeze method).
 +
 +
<br>07/08/2015:
 +
 +
- <br>Streaked chloramphenicol plates with transformed RFP DH5α Z1 cells.
 +
 +
- <br>Re-streaked plates with BclA and pgsA transformed cells.
 +
 +
- <br>Made chemically competent DH5 α Z1 cells.
 +
 +
- <br>Ran gel of digested plasmid. Obtained single band at 2000 base pairs (where
 +
 +
backbone without RFP would be, lacking band at 1000 base pairs (for RFP gene)).
 +
 +
- <br>Ran gel of undigested plasmids, single band at 3000 base pairs.
 +
 +
- <br>Gel purified digested plasmid, nanodropped (2.1ng/l).
 +
 +
<br>08/08/2015:
 +
 +
- <br>RFP and BclA transformed cells grew with single colonies.
 +
 +
- <br>pgsA plate showed no growth.
 +
 +
- <br>Inoculated falcon tubes with RFP and BclA.
 +
 +
- <br>Inoculated tube using old pgsA.
 +
 +
- <br>Plated pure DH5α Z1 onto 1 chloramphenicol and 1 clean plate (to establish
 +
 +
background growth).
 +
 +
<br>09/08/2015:
 +
 +
- <br>Mini prepped RFP, BclA and pgsA transformed cells.
 +
 +
- <br>Chloramphenicol plate grew no colonies, clean plate grew a lawn.
 
</p>
 
</p>
 
 
Line 270: Line 300:
 
<p>
 
<p>
  
−
<br>10/8/15
+
<br>10/08/2015:
  
−
<br>Nanodropped miniprep of RFP, BCLA and PGSA.
+
- <br>Nanodropped mini prep of RFP, BclA and pgsA.
−
Sent off for sequencing.  
+
  
−
<br>11/8/15
+
- <br>Sent off for sequencing.
  
−
<br>Digested old EC1 plasmid using Pst1, EcoR1 and Age1, Nde1 to obtain plasmid backbone and full construct … anchor proteins.
+
<br>11/08/2015
−
Digest new RFP plasmid to obtain plasmid backbone.
+
−
Inoculated culture with RFP bacteria, need to mini-prep tomorrow.
+
−
PCR if sZF10.
+
−
Mini-prep of RFP plasmid (JO4450).
+
  
−
<br>12/8/15
+
- <br>Digested old EC1 plasmid using Pst1+EcoR1 and Age1+Nde1 (to obtain
  
−
<br>Prep of 2 binding slides (overnight shaking 37oC, 6 hours 60oC oven, turned halfway) and 1 control (HCL bath only).
+
plasmid backbone, full construct and anchor proteins).
−
Miniprep new RFP plasmid, digest, gelled.
+
−
Gel of old full construct digested by Age1 and Pst1, gel extracted, nanodropped.
+
−
Gel extraction didn’t work.
+
−
Digestion of all zinc fingers.
+
  
−
<br>13/8/15
+
- <br>Digested new RFP plasmid to obtain plasmid backbone.
  
−
<br>Ligation of lpp-full construct into PSB1C3 backbone.
+
- <br>Inoculated culture with RFP bacteria.
−
Transformation of ligation product.
+
  
 +
- <br>PCR of sZF10.
  
 +
- <br>Mini-prep of RFP plasmid (JO4450).
  
−
<br>14/8/15
+
<br>12/08/2015
  
−
<br>Restriction digestion of full construct using Pst1 and EcoR1.
+
- <br>Prepared 2 binding glass slides and 1 control.
−
Inoculation of full construct transformed plasmid.
+
−
Only 2 colonies on the 4 plates of bacteria grew, background growth is 0 via control plates.  
+
  
−
<br>15/8/15
+
- <br>Mini prepped new RFP plasmid.
  
−
<br>Mini-prepped grown transformed DH5 Z1, colonies 1 and 2.
+
- <br>Digested new RFP plasmid and ran it on a gel.  
−
Plated normal DH5 Z1 on clean plates, found lawn, viable cells.
+
−
Re-transformed cells formed a lawn, taken to be re-spread, will inoculate tomorrow.
+
  
 +
- <br>Gel of old full construct digested by Age1+Pst1.
 +
 +
- <br>Gel extracted then nanodropped. Showed that the gel extraction didn’t work.
 +
 +
- <br>Digestion of all zinc fingers.
 +
 +
<br>13/08/2015
 +
 +
- <br>Ligation of Lpp OmpA full construct into pSB1C3backbone.
 +
 +
- <br>Transformation of ligation product.
 +
 +
<br>14/08/2015
 +
 +
- <br>Restriction digestion of Lpp OmpA full construct using Pst1+EcoR1.
 +
 +
- <br>Inoculation of Lpp OmpA full construct transformed plasmid.
 +
 +
- <br>Only 2 colonies on the 4 plates of bacteria grew (background growth is 0 via
 +
 +
control plates).
 +
 +
<br>15/08/2015
 +
 +
- <br>Mini-prepped grown transformed DH5α Z1, colonies 1 and 2.
 +
 +
- <br>Plated normal DH5α Z1 on clean plates (found a lawn of viable cells).
 +
 +
- <br>Re-transformed cells formed a lawn (taken to be re-spread and inoculated).
  
  
Line 329: Line 376:
 
<p>
 
<p>
  
−
17/8/15
+
<br>17/08/2015:
 +
 
 +
- <br>Prepared mini prep for sequencing.
 +
 
 +
- <br>Digested Lpp OmpA out of the plasmid and gel extracted the backbone.
 +
 
 +
- <br>Ligated all anchor proteins into the plasmid.
 +
 
 +
<br>18/08/2015:
 +
 
 +
- <br>Transformed DH5α Z1 cells with each anchor protein.
 +
 
 +
- <br>Re-growing Lpp OmpA transformed DH5α Z1 cells.
 +
 
 +
- <br>Electroporated MG1655Z1 cells using the original full construct plasmid and
 +
 
 +
plated.
 +
 
 +
- <br>Streaked FIM(negative) cells on a clean plate.
 +
 
 +
<br>19/08/2015:
 +
 
 +
- <br>All plated grew well (Lpp OmpA transformed cells formed a lawn).
 +
 
 +
- <br>Streaked Lpp OmpA transformed cells onto a chloramphenicol plate.
 +
 
 +
- <br>Electroporated cells did not grow.
 +
 
 +
- <br>Inoculations were made of the BclA, pgsA and INP plates.
 +
 
 +
- <br>Made 12 new chloramphenicol plates.
 +
 
 +
- <br>Designed primers (for the DNA origami oligonucleotide adhesive) for the
 +
 
 +
modellers.
 +
 
 +
- <br>Sequences came back clean (with no mutations).
 +
 
 +
- <br>Colony PCR of all grown cells.
 +
 
 +
<br>20/08/2015:
 +
 
 +
- <br>Ran an electrophoresis gel of the colony PCRs.
 +
 
 +
- <br>Made chemically competent FIM(negative).
 +
 
 +
- <br>Made glycerol stocks of all completed cell cultures.
 +
 
 +
- <br>Mini prep of pgsA, BclA and INP plasmids.
 +
 
 +
- <br>Made inoculations of Lpp OmpA (one with IPTG, one without, and one where
 +
 
 +
IPTG is added later).
 +
 
 +
<br>21/08/2015:
 +
 
 +
- <br>BclA, INP and pgsA plasmids sent for sequencing.
 +
 
 +
- <br>Finished making FIM(negative) chemically competent cells.
 +
 
 +
- <br>Measured OD600 of Lpp OmpA inoculations.
 +
 
 +
- <br>Made another Lpp OmpA inoculation.
 +
 
 +
- <br>Remade the colony PCR.
 +
 
 +
<br>22/08/2015:
 +
 
 +
- <br>Ran electrophoresis gel of colony PCR.
  
−
<br>Prepared miniprep for sequencing.
+
- <br>No results so colony PCR remade.
−
<br>Digested the lpp out of the plasmid and gel extracted the backbone.
+
−
<br>Ligated all anchor proteins into the plasmid.  
+
  
−
<br>18/8/15
+
<br>23/08/2015
  
−
<br>Transformed DH5 Z1 cells with each anchor protein.
+
- <br>No results.
−
<br>Re-growing lpp transformed DH5 Z1 cells, plan to make glycerol stock later.
+
−
<br>Electroporated MG1655Z1 cells using the original full construct plasmid.
+
−
<br>Plated/streaked FIM(negative) cells on a clean plate.
+
  
−
<br>19/8/15
+
- <br>Made inoculations of Lpp OmpA transformed cells (with IPTG and normal DH5α
  
−
<br>All plated grew well, lpp transformed cells formed lawn.
+
Z1 cells).
−
<br>Streaked lpp transformed cells.
+
−
<br>Electroporated cells did not grow.
+
−
<br>Inoculations were made of the BCLA, PGSA and INP plates.
+
−
<br>Made 12 new chlor plates.
+
−
<br>Designed primers for the modellers.
+
−
<br>Sequences came back with no mutations!
+
−
<br>Colony PCR of all grown cells, will run on gel tomorrow.  
+
 
</p>
 
</p>
 
 
Line 372: Line 475:
  
  
 +
<br>24/08/2015:
  
 +
- <br>Made colony PCR again using different primer-colony combinations.
 +
 +
- <br>Made more chloramphenicol plates.
 +
 +
- <br>Assembled primers.
 +
 +
- <br>Made designs (using paper stencils) of RFP transformed DH5α Z1.
 +
 +
- <br>Carried out Age1+Nde1 digestion of Lpp OmpA full construct.
 +
 +
- <br>Ran electrophoresis gel of colony PCR – had negative results.
 +
 +
<br>25/08/2015:
 +
 +
- <br>Gel extraction of digested full construct plasmid showed no positive results.
 +
 +
- <br>Mini prepped functional cell.
 +
 +
- <br>Set up (8 hour) digestion.
 +
 +
<br>26/08/2015:
 +
 +
- <br>Analyse digestion. Showed a single band (a negative result).
 +
 +
- <br>Put DH5αcells on to grow (for microscopy).
 +
 +
<br>27/08/2015:
 +
 +
- <br>First steps of slide preparation.
 +
 +
- <br>Mini prepped Lpp OmpA full construct.
 +
 +
- <br>Digested Lpp OmpA full construct with Age1.
 +
 +
- <br>Ran an electrophoresis gel of the digestion - results were negative.
 +
 +
<br>28/08/2015:
 +
 +
- <br>Mini prepped Lpp OmpA full construct.
 +
 +
- <br>Ran electrophoresis gel (of digestion with EcoR1+Nde1 and digestion with
 +
 +
EcoR1+Age1).
 +
 +
- <br>Rerunning electrophoresis gel (using old enzymes and Lpp OmpA plasmid full
 +
 +
construct mini prep).
 +
 +
<br>29/08/2015:
 +
 +
- <br>Did gel extraction of Lpp OmpA full construct plasmid mini prep.
 +
 +
- <br>No supernatant after freeze and squeeze step.
 +
 +
- <br>MG1655Z1 cells transformed with PSB3K3 via electroporation.
 +
 +
<br>30/08/2015:
 +
 +
- <br>Redid gel extraction of Lpp OmpA full construct (plasmid mini prep) via.
  
  
Line 395: Line 558:
  
  
 +
<br>31/08/2015:
  
 +
- <br>Repeated gel extraction (not enough full construct mini prepped plasmid).
 +
 +
- <br>Started mini prep of Lpp OmpA full construct transformed colony.
 +
 +
- <br>Slide prep up to (but not including) Blocking Buffer wash.
 +
 +
- <br>LB inoculated with colony from MG1655Z1 + pSB3K3 plate.
 +
 +
<br>01/09/2015
 +
 +
- <br>Gel extracted anchor protein plasmids (from digestion).
 +
 +
- <br>Microscopy session - some controls not the best, but significant difference in
 +
 +
brightness of induced and Lpp OmpA full construct.
 +
 +
<br>02/09/2015:
 +
 +
- <br>PCR of sZFs 2, sZF10 sZFand 14.
 +
 +
- <br>Mini prepped more plasmid from the working MG1655Z1cells.
 +
 +
- <br>Set up more MG1655Z1cells to grow overnight.
 +
 +
- <br>Restriction digestion of full construct plasmid carried out using Nhe1+Cla1.
 +
 +
<br>03/09/2015:
 +
 +
- <br>Triplicate mini prep made using colony 2 (MG1655Z1).
 +
 +
- <br>Restriction digested mini prep.
 +
 +
- <br>Ran a gel extraction of digestion, then extracted from gel.
 +
 +
- <br>Ran a PCR of the cut zinc fingers.
 +
 +
<br>04/09/2015:
 +
 +
- <br>Mini prepped a large batch of Dh5α Z1cell.
 +
 +
- <br>Ran a restriction digestion (using Age1+Nde1).
 +
 +
- <br>JS006 transformed with PSB3K3 via electroporation, grown on 1:2000
 +
 +
Kanamycin plate.
 +
 +
- <br>Successful PCR of sZF2 and sZF14 (purified and tested).
 +
 +
- <br>Redid PCR of sZF10.
 +
 +
- <br>Gel extraction of plasmid Mhe1+Cla1 digest, worked perfectly.
 +
 +
- <br>PCR purified the 3 anchor proteins (BclA, pgsA and INP).
 +
 +
- <br>Mini prepped anchor proteins.
 +
 +
- <br>Digested the anchor proteins, ran on an electrophoresis gel and then extracted.
 +
 +
- <br>Carried out a restriction digestion (with Age1+Nde1).
 +
 +
- <br>Ran digestion on an electrophoresis gel, and then extracted.
 +
 +
<br>05/09/2015
 +
 +
- <br>Gel extracted the plasmid digested by Nhe1+Cla1.
 +
 +
- <br>Mini prepped more plasmid.
 +
 +
- <br>Digestion of plasmid (using Nde1+Age1).
 +
 +
- <br>Ran electrophoresis gel of Nde1+Age1 digestion.
  
  
Line 414: Line 649:
  
  
 +
<br>07/09/2015:
  
 +
- <br>Ran an electrophoresis gel of Nde1+Age1 digest (reran to separate out bands).
 +
 +
- <br>Gel extracted.
 +
 +
- <br>Digested all zinc fingers.
 +
 +
- <br>Gel of full construct (digested with Nhe1+Cla1) showed a positive result, so PCR
 +
 +
purified.
 +
 +
- <br>PCR purified sZF2, sZF10 and sZF14.
 +
 +
- <br>Ligated (using T4 DNA ligase protocol).
 +
 +
<br>08/09/2015:
 +
 +
- <br>Ran gel electrophoresis of ligated zinc fingers (to check them).
 +
 +
- <br>Transformed some of the ligation into chemically competent DH5α Z1 cells.
 +
 +
- <br>Transformed rest of the ligation into electrocompetent cells.
 +
 +
- <br>Made more chloramphenicol plates.
 +
 +
<br>09/09/2015:
 +
 +
- <br>Primer assembly of fluorescent zinc finger specific binding sequences.
 +
 +
- <br>Made oligonucleotides for glass DNA binding.
 +
 +
- <br>Made fluorescent oligos for specific DNA binding test.
 +
 +
<br>10/09/2015:
 +
 +
- <br>Plasmid preparation of pSB3K3 for insertion of fluorescent gBlocks.
 +
 +
- <br>Wells 6F/18A/18C/18E/18E from kit Plate 4 2015 Phusion PCR.
 +
 +
- <br>Gel electrophoresis of PCR products - showed only primer dimer at 100 bp.
 +
 +
<br>11/09/2015:
 +
 +
- <br>Plasmid preparation of pSB3K3 for insertion of fluorescent gBlocks.
 +
 +
- <br>Wells 6F/18A/18C/18E/18E from kit Plate 4 2015 Phusion PCR.
 +
 +
- <br>Gel electrophoresis of PCR products - showed only primer dimer at 100 base
 +
 +
pairs again.
 +
 +
- <br>Mini prepped electroporated MG1655Z1 containing sZFf2, sZF10, sZF14, BclA
 +
 +
and INP plasmids.
 +
 +
- <br>Sent the 5 modified plasmids for sequencing.
 +
 +
- <br>Received PCR product from Oxford iGEM. Assembled oligos in PCR machine.  
 +
 +
- <br>Prepared slides (of induced and uninduced zinc fingers) for microscopy using
 +
 +
Dh5α Z1 cells.
 +
 +
<br>12/09/2015:
 +
 +
- <br>Made inoculations of MG1655Z1 transformed cells for preparation of slides
 +
 +
(using induced and uninduced zinc fingers and anchors).
  
  
Line 437: Line 740:
  
  
 +
<br>14/09/2015
 +
 +
- <br>Redo Phusion PCR (gradient) of wells 6F/18A/18C/18E/18E from kit Plate 4
 +
 +
2015 with +/- DMSO.
 +
 +
- <br>Gel electrophoresis of PCR products identified band of 2.5kb.
 +
 +
- <br>Dpn1 digest for 3 hours and 37°C
 +
 +
- <br>Remade glass slides with all zinc fingers (for the oligo binding experiment –
 +
 +
experiment 3) and anchor proteins (for FLAG tag experiment – experiment 2).
 +
 +
<br>15/09/2015
 +
 +
- <br>PCR purification of 18C and 18E.
 +
 +
- <br>Gibson assembly of 18C and 18E, each with the gBlock for mKate, GFP and
 +
 +
Cerulean
 +
 +
- <br>Remade slides with zinc fingers (for experiment 3) and anchor proteins
 +
 +
(experiment 2). Found out later that sequencing did not come back right, so slides
 +
 +
must be remade using new cells. .
 +
 +
<br>16/09/2015
 +
 +
- <br>Grew up all zinc fingers cells in m9 (to be used for experiment 2). 
 +
 +
<br>17/09/2015
 +
 +
- <br>Cells had not grown up, so regrew them in LB.
 +
 +
- <br>Once grown, these cells were used to prepare slides for experiment 2.
 +
 +
<br> 18/09/2015
 +
 +
<br> Visualised slides of zinc finger transformed cells, but only got negative results.
  
 +
<br> Remade slides using alternative protocol (did not fix cells to slide, cells were in liquid media). Visualised, got positive results for sZF14.
  
  

Latest revision as of 20:11, 18 September 2015

Warwick iGEM 2015

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