Team:Hawaii/Notebook/2008-07-28

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:*The omega region from pSMC121 and the rep/mob region are giving primer dimers and misamplification respectively. I am trying a gradient PCR from 49°C to 55°C to see if this will fix the problem.
:*The omega region from pSMC121 and the rep/mob region are giving primer dimers and misamplification respectively. I am trying a gradient PCR from 49°C to 55°C to see if this will fix the problem.
 +
=== PCR for BB-pRL1383a construction/verification===
 +
:<strong>Grace</strong>
 +
 +
:* PCR of BB-pRL1383a (from plasmid prep) using VF2 & VR_B0034 primers to verify B0034 insert and MCS replacement
 +
:* PCR of B0034 with HindIII and BamHI primers in case we need to redo BB-pRL1383a construction
 +
::* Maintained everything  on ice this time
 +
::* Used 0.5&mu;l plasmid in PCR reaction
 +
::* Annealing at 55C
 +
::* Extension for 30 sec. at 72C
 +
 +
===Plasmid preps===
 +
:<strong>Grace</strong>
 +
 +
:* Resuspended plasmid preps in 30 &mu;l TE and checked concentrations on nanodrop spectrometer
 +
::* nir+B0034 = 13.6 ng/&mu;l
 +
::* GFP+B0024 = 9 ng/&mu;l
 +
::* GFPf+B0024= 7.1 ng/&mu;l
 +
::* BB-pRL1383a = 166.3 ng/&mu;l
 +
:* Restriction digested nir+B0034 plasmid prep to verify insert (GFP and GFPf constructs were verified last week)
 +
::* Digested w/ EcoRI and SpeI so we can extract from gel if insert is correct
 +
::* Incubated at 37C for 2 hours
 +
 +
===GFP(f) + tt constructs===
 +
:<strong>Grace</strong>
 +
 +
:* Restriction digested GFP and GFPf w/ EcoRI and SpeI in 50 &mu;l reactions for 3 hours
 +
:* Restriction digested B0024 (tt) with XbaI then EcoRI for 3 hours total
 +
:* Ran on an EtBr stained 3.8% agarose gel alongside GFP+B0024 ligation from 7/22 to see if ligation was successful (and the problem was with the colony we picked)
 +
:* Gel purified bands and ligated GFP(f) with B0024
 +
::* Ligation reaction incubated at room temperature for 2 hours.
 +
 +
===Transformation of GFP(f)+tt constructs into DH5&alpha;===
 +
:<strong>Krystle</strong>
== Drylab Work ==
== Drylab Work ==

Revision as of 23:11, 28 July 2008

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Notebook (t) Meetings (t)

Things we did today

Wetlab work

Transformation

Margaret
  • Transformation of I14032 into Db3.1

Colony PCR

Margaret
  • Verifying the inserts of two I51020 (the base vector) and one colony of pSB1A7 (an insulated vector with high copy #)

PCR amplification of pRL1383a

Margaret
  • The omega region from pSMC121 and the rep/mob region are giving primer dimers and misamplification respectively. I am trying a gradient PCR from 49°C to 55°C to see if this will fix the problem.

PCR for BB-pRL1383a construction/verification

Grace
  • PCR of BB-pRL1383a (from plasmid prep) using VF2 & VR_B0034 primers to verify B0034 insert and MCS replacement
  • PCR of B0034 with HindIII and BamHI primers in case we need to redo BB-pRL1383a construction
  • Maintained everything on ice this time
  • Used 0.5μl plasmid in PCR reaction
  • Annealing at 55C
  • Extension for 30 sec. at 72C

Plasmid preps

Grace
  • Resuspended plasmid preps in 30 μl TE and checked concentrations on nanodrop spectrometer
  • nir+B0034 = 13.6 ng/μl
  • GFP+B0024 = 9 ng/μl
  • GFPf+B0024= 7.1 ng/μl
  • BB-pRL1383a = 166.3 ng/μl
  • Restriction digested nir+B0034 plasmid prep to verify insert (GFP and GFPf constructs were verified last week)
  • Digested w/ EcoRI and SpeI so we can extract from gel if insert is correct
  • Incubated at 37C for 2 hours

GFP(f) + tt constructs

Grace
  • Restriction digested GFP and GFPf w/ EcoRI and SpeI in 50 μl reactions for 3 hours
  • Restriction digested B0024 (tt) with XbaI then EcoRI for 3 hours total
  • Ran on an EtBr stained 3.8% agarose gel alongside GFP+B0024 ligation from 7/22 to see if ligation was successful (and the problem was with the colony we picked)
  • Gel purified bands and ligated GFP(f) with B0024
  • Ligation reaction incubated at room temperature for 2 hours.

Transformation of GFP(f)+tt constructs into DH5α

Krystle

Drylab Work

Primer Design

Margaret
  • I want to design primers for some replication proteins + origin from pSB2K3

Discussion

Quote of the Day

History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson


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