User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/06/29: Difference between revisions

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* Need to design new primers (regular and mutagenic) for amplifying PHIX174 genome.
* Need to design new primers (regular and mutagenic) for amplifying PHIX174 genome.


__NOTOC__
==To do==
* Today
** [http://openwetware.org/wiki/Corum:DNA_Hybridization Hydridize] SphI-PA-UTR1-NcoI, SphI-PD-UTR1-NcoI linker, and SphI-PF-UTR1-NcoI linker to 1mM stock concentration. Serial dilutions 1/10 to 100 nM final concentration.
** To create the following linkers:
**# SphI-PB-UTR1-NcoI linker
**# SphI-PG-UTR1-NcoI linker
**# SphI-PL-UTR1-NcoI linker
**# SphI-PLPA-UTRA-NheI
*** Perform [http://openwetware.org/wiki/Corum:PCR PCR] using ΦX174 genomic DNA as template.
*** Follow with [http://openwetware.org/wiki/Corum:PCR_Purification PCR purification]. Elute with 100 μL H<sub>2</sub>O and speedvac to ~20 μL.
*** [http://openwetware.org/wiki/Corum:Digestion Double digest] overnight with SphI and NcoI (except SphI-PLPA-UTRA-NheI, which of course requires SphI and NcoI).
** [http://openwetware.org/wiki/Corum:T4_Ligation Ligate] 2.5 μL pBEST-SphI//NheI-deGFP-T500 (2nM) with 5 μL linkers SphI-PX-UTRX-NheI (X = null, A, B, D, F, G, L; ~100nM) to create the following constructs:
**# pBEST-null-deGFP-T500
**# pBEST-PA-UTRA-deGFP-T500
**# pBEST-PB-UTB-null-deGFP-T500
**# pBEST-PD-UTRD-null-deGFP-T500
**# pBEST-PF-UTRF-null-deGFP-T500
**# pBEST-PG-UTRG-null-deGFP-T500
**# pBEST-PL-UTRL-null-deGFP-T500
 
* Today +1
** To obtain following linkers, perform [http://openwetware.org/wiki/Corum:Gel_Purification Gel extraction] (elute in 100 μL H<sub>2</sub>O, linkers should be at ~100nM concentration):
**# SphI-PB-UTR1-NcoI linker
**# SphI-PG-UTR1-NcoI linker
**# SphI-PL-UTR1-NcoI linker
**# SphI-PLPA-UTRA-NheI
** [http://openwetware.org/wiki/Corum:T4_Ligation Ligate] 0.5 μL pBEST-SphI//NcoI-UTR1-deGFP-T500 (2nM) with 5 μL linkers SphI-PX-UTRX-NheI (X = B, G, L, LA; ~100nM) to create the following constructs:
**# pBEST-PB-UTR1-deGFP-T500
**# pBEST-PG-UTR1-deGFP-T500
**# pBEST-PL-UTR1-deGFP-T500
**# pBEST-PLPA-UTR1-deGFP-T500
** [http://openwetware.org/wiki/Corum:T4_Ligation Ligate] 2.5 μL pBEST-SphI//NheI-deGFP-T500 (2nM) with 5 μL linker SphI-PLPA-UTRX-NheI to create the following construct:
**# pBEST-PLPA-UTR1-deGFP-T500
 
* Today +2
** [http://openwetware.org/wiki/Corum:Transformation Transform] the following ligation products into JM109 competent cells and grow overnight.
**# pBEST-PA-UTR1-deGFP-T500
**# pBEST-PB-UTR1-deGFP-T500
**# pBEST-PD-UTR1-deGFP-T500
**# pBEST-PF-UTR1-deGFP-T500
**# pBEST-PG-UTR1-deGFP-T500
**# pBEST-PL-UTR1-deGFP-T500
**# pBEST-PLPA-UTR1-deGFP-T500
**# pBEST-PA-UTRA-deGFP-T500
**# pBEST-PB-UTRB-deGFP-T500
**# pBEST-PD-UTRD-deGFP-T500
**# pBEST-PF-UTRF-deGFP-T500
**# pBEST-PG-UTRG-deGFP-T500
**# pBEST-PL-UTRL-deGFP-T500
**# pBEST-PLPA-UTRA-deGFP-T500
**# pBEST-NULL-deGFP-T500
 
* Today +3
** Select 4 colonies per plate and grow 2.5 mL miniculures (4 × 15 = 60 total).


==To do==
* Today +4&5
** [http://openwetware.org/index.php?title=Corum:Miniprep Miniprep] the minicultures. Submit samples for [http://openwetware.org/wiki/Corum:DNA_Sequencing sequencing].


__NOTOC__


==Notes==
==Notes==

Revision as of 11:18, 29 June 2012

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Characterization B: Expression of PHIX174 promoters fused to UTR1-deGFP.

  • I peformed hybridization on oligonucleotides to construct the following:
    • SphI-PA-UTR1-NcoI linker at 100 nM final concentration
    • SphI-PD-UTR1-NcoI linker at 100 nM final concentration
    • SphI-PF-UTR1-NcoI linker at 100 nM final concentration
  • I peformed PCR followed by PCR purification, speedvac down to ~20 μL, digestion with SphI and NcoI, and another round of PCR purification to create the following linkers:
    • SphI-PB-UTR1-NcoI linker
    • SphI-PG-UTR1-NcoI linker
    • SphI-PL-UTR1-NcoI linker
    • SphI-PLPA-UTR1-NcoI linker
  • All of the linkers should be at ~100 nM final concentration, so they can be used with the pBEST-SphI//NcoI-deGFP-T500 linker (10 nM) in ligation to create the desired set of pBEST-PX-UTR1-deGFP-T500 constructs.

Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.

  • There were no colonies after transformation. Therefore, I decided to characterize all linkers and backbones for those constructs as well as the UTR1-deGFP constructs via quantifluore DNA quantification. Results were as follows:
    • SphI-null-NheI linker = 73 nM
    • SphI-PA-UTRA-NheI linker = 46 nM
    • SphI-PB-UTRB-NheI linker = 37 nM
    • SphI-PD-UTRD-NheI linker = 66 nM
    • SphI-PF-UTRF-NheI linker = 66 nM
    • SphI-PG-UTRG-NheI linker = 139 nM
    • SphI-PL-UTRL-NheI linker = 87 nM
    • SphI-PLPA-UTRA-NheI raw PCR product = 166 nM
    • pBEST-SphI//NheI -deGFP-T500 backbone = 2 nM
  • All of the linkers are ~100 nM, which is what is expected for proper ligation.
  • The backbone is at expected 2 nM concentration but is needed at ~10 nM concentration, so to obtain a proper ligation with 1:100 backbone to linker concentration, the volume of backbone needs to be increased by a factor of 5 in ligation. I performed ligation with 2.5 μL backbone and 5 μL linkers.
    • I digested and then PCR purified the SphI-PLPA-UTRA-NheI raw PCR product with SphI and NheI overnight to create the SphI-PLPA-UTRA-NheI linker (yet again)

Hypothesis 2: Gene L is necessary for phage propagation.

  • Need to design new primers (regular and mutagenic) for amplifying PHIX174 genome.

To do

  • Today
    • Hydridize SphI-PA-UTR1-NcoI, SphI-PD-UTR1-NcoI linker, and SphI-PF-UTR1-NcoI linker to 1mM stock concentration. Serial dilutions 1/10 to 100 nM final concentration.
    • To create the following linkers:
      1. SphI-PB-UTR1-NcoI linker
      2. SphI-PG-UTR1-NcoI linker
      3. SphI-PL-UTR1-NcoI linker
      4. SphI-PLPA-UTRA-NheI
      • Perform PCR using ΦX174 genomic DNA as template.
      • Follow with PCR purification. Elute with 100 μL H2O and speedvac to ~20 μL.
      • Double digest overnight with SphI and NcoI (except SphI-PLPA-UTRA-NheI, which of course requires SphI and NcoI).
    • Ligate 2.5 μL pBEST-SphI//NheI-deGFP-T500 (2nM) with 5 μL linkers SphI-PX-UTRX-NheI (X = null, A, B, D, F, G, L; ~100nM) to create the following constructs:
      1. pBEST-null-deGFP-T500
      2. pBEST-PA-UTRA-deGFP-T500
      3. pBEST-PB-UTB-null-deGFP-T500
      4. pBEST-PD-UTRD-null-deGFP-T500
      5. pBEST-PF-UTRF-null-deGFP-T500
      6. pBEST-PG-UTRG-null-deGFP-T500
      7. pBEST-PL-UTRL-null-deGFP-T500
  • Today +1
    • To obtain following linkers, perform Gel extraction (elute in 100 μL H2O, linkers should be at ~100nM concentration):
      1. SphI-PB-UTR1-NcoI linker
      2. SphI-PG-UTR1-NcoI linker
      3. SphI-PL-UTR1-NcoI linker
      4. SphI-PLPA-UTRA-NheI
    • Ligate 0.5 μL pBEST-SphI//NcoI-UTR1-deGFP-T500 (2nM) with 5 μL linkers SphI-PX-UTRX-NheI (X = B, G, L, LA; ~100nM) to create the following constructs:
      1. pBEST-PB-UTR1-deGFP-T500
      2. pBEST-PG-UTR1-deGFP-T500
      3. pBEST-PL-UTR1-deGFP-T500
      4. pBEST-PLPA-UTR1-deGFP-T500
    • Ligate 2.5 μL pBEST-SphI//NheI-deGFP-T500 (2nM) with 5 μL linker SphI-PLPA-UTRX-NheI to create the following construct:
      1. pBEST-PLPA-UTR1-deGFP-T500
  • Today +2
    • Transform the following ligation products into JM109 competent cells and grow overnight.
      1. pBEST-PA-UTR1-deGFP-T500
      2. pBEST-PB-UTR1-deGFP-T500
      3. pBEST-PD-UTR1-deGFP-T500
      4. pBEST-PF-UTR1-deGFP-T500
      5. pBEST-PG-UTR1-deGFP-T500
      6. pBEST-PL-UTR1-deGFP-T500
      7. pBEST-PLPA-UTR1-deGFP-T500
      8. pBEST-PA-UTRA-deGFP-T500
      9. pBEST-PB-UTRB-deGFP-T500
      10. pBEST-PD-UTRD-deGFP-T500
      11. pBEST-PF-UTRF-deGFP-T500
      12. pBEST-PG-UTRG-deGFP-T500
      13. pBEST-PL-UTRL-deGFP-T500
      14. pBEST-PLPA-UTRA-deGFP-T500
      15. pBEST-NULL-deGFP-T500
  • Today +3
    • Select 4 colonies per plate and grow 2.5 mL miniculures (4 × 15 = 60 total).


Notes

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Digital Signature

  • SC 11:50, 29 June 2012 (EDT):
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