Team:British Columbia/Notebook/CRISPR

From 2013.igem.org

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Target:
Target:
*tracerRNA
*tracerRNA
-
*repeat + spacer + repeat - Vanins*2 + underlings
+
*repeat + spacer + repeat -  
*leader - Ray, Fisal, Dan
*leader - Ray, Fisal, Dan
Decoy:
Decoy:
*PAM + spacer (on Amp)
*PAM + spacer (on Amp)
 +
 +
 +
==='''July 4th'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Phosphorylate and assemble T4, T7, and tracrRNA ordered oligos for decoy plasmids.
 +
 +
'''Results:''' Oligonucleotides were assembled using this protocol.
 +
 +
 +
==='''July 5th'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Ligated annealed oligos into PSB1C3, cut with E and S, and transform into 10G  heat competent cells. Cells were recovered in 300uL and 25uL wa plated along with a no insert control.
 +
 +
'''Results:'''  NA
 +
 +
 +
==='''July 6th'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Select colonies for screening
 +
 +
'''Results:''' Approximately 100 colonies were present for both the T4, T7 and tracr RNA spacers with approximately 10 colonies on the background plate. Colony PCR with VF and VR2 confirmed bands at approximately 350bp. 3 colonies from both T4, T7 and tracrRNA plates were sent for Sanger sequencing through genewiz.
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==='''July 9th'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Phosphorylate, ligate and amplify oligos for T4 and T7 repeat-spacer arrays.
 +
 +
'''Results:''' The T4 and T7 spacers, repeat and the Repeat-BB Suffix oligonucleotides were phosphorylated with T4 PNK.
 +
 +
All oligonucleotides were mixed in equimolar amounts at a final concentration of 10 nM each, annealed and ligated and PCR amplified according to the repeat-assembly protocol.
 +
==='''July 10th'''===
==='''July 10th'''===
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'''Results:''' Unsuccessful no bands were seen on the agarose gel.
'''Results:''' Unsuccessful no bands were seen on the agarose gel.
 +
==='''July 10th'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Gel purify assembled sequences.
 +
 +
'''Results:''' The PCR product from yesterday was loaded into a 10% TBE-PAGE gel and run at 100V until the blue marker ran off. Bands corresponding to one (148 bp) and two (219 bp) were excised and purified.
 +
 +
==='''July 11th'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Finish purification, cut, ligate and transform repeat-spacer assemblies
 +
 +
'''Results:''' Gel slurries were filtered and ethanol precipitated and quantified. Products were cut with E+S, ligated into PSB1C3 and transformed.
 +
 +
==='''July 12th'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Finish purification, cut, ligate and transform repeat-spacer assemblies
 +
 +
'''Results:''' Gel slurries were filtered and ethanol precipitated and quantified. Products were cut with E+S, ligated into PSB1C3 and transformed.
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==='''July 15th'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Screen/confirm insert length.
 +
 +
'''Results:''' 5 colonies from the single insert and 10 colonies from the double insert plate were screened via colony PCR for correct insert length. Three positive colonies from the single insert and 6 positive colonies from the double insert were inoculated for overnight cultures.
 +
 +
==='''July 17th'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Figure out why no T7 sequences were assembled.
 +
 +
'''Results:''' Sequencing results showed that the T4 spacer was correctly assembled into the repeat-spacer-arrays for single and double inserts. Unfortunately, no T7 spacers were incorporated in the assembly. After going over the sequence of the oligonucleotides with Mike, this is most likely because one of the T7 splint sequences was not correctly designed; we accidentally left the PAM site in it (which would have prevented ligation). The splint was redesigned and reordered; also designed and ordered a second set of different T4 and T7 spacers.
 +
 +
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==='''July 22nd'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Restart assembly with fixed splint
 +
 +
'''Results:''' The repeat-spacer assembly was restarted with the fixed T7 splint and the new spacers and splints. New splints were phosphorylated with T4 PNK.
 +
 +
All oligonucleotides were mixed in equimolar amounts at a final concentration of 10 nM each, annealed and ligated and PCR amplified according to the repeat-assembly protocol.
 +
 +
 +
==='''July 23rd'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Gel purify assembled sequences.
 +
 +
'''Results:''' The PCR product from yesterday was loaded into a 10% TBE-PAGE gel and run at 100V until the bromophenol blue marker ran off. Bands corresponding to one (148 bp) and two (219 bp) were excised and purified.
 +
 +
==='''July 24th'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Cut, ligate and transform repeat-spacer assemblies
 +
 +
'''Results:''' Gel purification products were quantified with the Qubit. Single inserts for T7 and the new T4 and T7, and double inserts for T4+T7 (original set) were cut with E+S, ligated into PSB1C3 and transformed.
 +
 +
==='''July 25th'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Screen/confirm insert length.
 +
 +
'''Results:''' 5 colonies from each of the single insert and 10 colonies from the double insert plates were screened via colony PCR for correct insert length. Two positive colonies from each of the single insert and 6 positive colonies from each of the double inserts were inoculated for overnight cultures.
 +
 +
==='''July 26th'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Miniprep and prepare for sequencing
 +
 +
'''Results:''' Miniprepped overnight cultures from yesterday and send to sequence confirm.
 +
 +
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==='''July 29th'''===
 +
 +
'''Experimenter''': Cam Strachan
 +
 +
'''Aim:''' Analyze sequencing results
 +
 +
'''Results:''' Based on the sequencing results, the single insert T7, new T4 and T7 and the double T4 and T7 inserts were correctly assembled. The constructs were given to Joe to add promoters and a terminator via standard assembly.
 +
 +
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Revision as of 07:13, 27 September 2013

iGEM Home

Contents

Team CRISPR

July 3

List of BioBricks that are/will be on the way:

Cas9:

  • Cas9 - Joe and Anna
  • pTet -
  • const. promoter + Cas9 - Joe
  • promoter + Cas9 + Leader + R + spacer + R (on Kan)
  • L + R + spacer + R

Target:

  • tracerRNA
  • repeat + spacer + repeat -
  • leader - Ray, Fisal, Dan

Decoy:

  • PAM + spacer (on Amp)


July 4th

Experimenter: Cam Strachan

Aim: Phosphorylate and assemble T4, T7, and tracrRNA ordered oligos for decoy plasmids.

Results: Oligonucleotides were assembled using this protocol.


July 5th

Experimenter: Cam Strachan

Aim: Ligated annealed oligos into PSB1C3, cut with E and S, and transform into 10G heat competent cells. Cells were recovered in 300uL and 25uL wa plated along with a no insert control.

Results: NA


July 6th

Experimenter: Cam Strachan

Aim: Select colonies for screening

Results: Approximately 100 colonies were present for both the T4, T7 and tracr RNA spacers with approximately 10 colonies on the background plate. Colony PCR with VF and VR2 confirmed bands at approximately 350bp. 3 colonies from both T4, T7 and tracrRNA plates were sent for Sanger sequencing through genewiz.

July 9th

Experimenter: Cam Strachan

Aim: Phosphorylate, ligate and amplify oligos for T4 and T7 repeat-spacer arrays.

Results: The T4 and T7 spacers, repeat and the Repeat-BB Suffix oligonucleotides were phosphorylated with T4 PNK.

All oligonucleotides were mixed in equimolar amounts at a final concentration of 10 nM each, annealed and ligated and PCR amplified according to the repeat-assembly protocol.


July 10th

Experimenter: Anna Müller

Aim: PCR the cas9 gene from Streptococcus thermophilus cells.

Results: Unsuccessful no bands were seen on the agarose gel.

July 10th

Experimenter: Cam Strachan

Aim: Gel purify assembled sequences.

Results: The PCR product from yesterday was loaded into a 10% TBE-PAGE gel and run at 100V until the blue marker ran off. Bands corresponding to one (148 bp) and two (219 bp) were excised and purified.

July 11th

Experimenter: Cam Strachan

Aim: Finish purification, cut, ligate and transform repeat-spacer assemblies

Results: Gel slurries were filtered and ethanol precipitated and quantified. Products were cut with E+S, ligated into PSB1C3 and transformed.

July 12th

Experimenter: Cam Strachan

Aim: Finish purification, cut, ligate and transform repeat-spacer assemblies

Results: Gel slurries were filtered and ethanol precipitated and quantified. Products were cut with E+S, ligated into PSB1C3 and transformed.

July 15th

Experimenter: Cam Strachan

Aim: Screen/confirm insert length.

Results: 5 colonies from the single insert and 10 colonies from the double insert plate were screened via colony PCR for correct insert length. Three positive colonies from the single insert and 6 positive colonies from the double insert were inoculated for overnight cultures.

July 17th

Experimenter: Cam Strachan

Aim: Figure out why no T7 sequences were assembled.

Results: Sequencing results showed that the T4 spacer was correctly assembled into the repeat-spacer-arrays for single and double inserts. Unfortunately, no T7 spacers were incorporated in the assembly. After going over the sequence of the oligonucleotides with Mike, this is most likely because one of the T7 splint sequences was not correctly designed; we accidentally left the PAM site in it (which would have prevented ligation). The splint was redesigned and reordered; also designed and ordered a second set of different T4 and T7 spacers.


July 22nd

Experimenter: Cam Strachan

Aim: Restart assembly with fixed splint

Results: The repeat-spacer assembly was restarted with the fixed T7 splint and the new spacers and splints. New splints were phosphorylated with T4 PNK.

All oligonucleotides were mixed in equimolar amounts at a final concentration of 10 nM each, annealed and ligated and PCR amplified according to the repeat-assembly protocol.


July 23rd

Experimenter: Cam Strachan

Aim: Gel purify assembled sequences.

Results: The PCR product from yesterday was loaded into a 10% TBE-PAGE gel and run at 100V until the bromophenol blue marker ran off. Bands corresponding to one (148 bp) and two (219 bp) were excised and purified.

July 24th

Experimenter: Cam Strachan

Aim: Cut, ligate and transform repeat-spacer assemblies

Results: Gel purification products were quantified with the Qubit. Single inserts for T7 and the new T4 and T7, and double inserts for T4+T7 (original set) were cut with E+S, ligated into PSB1C3 and transformed.

July 25th

Experimenter: Cam Strachan

Aim: Screen/confirm insert length.

Results: 5 colonies from each of the single insert and 10 colonies from the double insert plates were screened via colony PCR for correct insert length. Two positive colonies from each of the single insert and 6 positive colonies from each of the double inserts were inoculated for overnight cultures.

July 26th

Experimenter: Cam Strachan

Aim: Miniprep and prepare for sequencing

Results: Miniprepped overnight cultures from yesterday and send to sequence confirm.


July 29th

Experimenter: Cam Strachan

Aim: Analyze sequencing results

Results: Based on the sequencing results, the single insert T7, new T4 and T7 and the double T4 and T7 inserts were correctly assembled. The constructs were given to Joe to add promoters and a terminator via standard assembly.


September 22nd

Experimenter: Anna Müller

Aim: Digest T4, T7 spacer sequence with PAM site(decoy construct) with EcoRI and SpeI to later on ligate into plasmid with ampicillin resistance.

Results: will follow.


Aim: Digest PsBIA3 plasmid with ampicillin resistance with EcoRI and SpeI to construct the decoy plasmides.

Results: will follow.


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