Difference between revisions of "Part:BBa K530028:Design"
(→‎Design Notes)
 
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Template DNA (~25ng) [1 uL from miniprep]
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'''Control reaction''' – this is your background! Add 50uL master mix (without primers) to PCR tube and put it on the cycler, identical to the other tubes. DpnI digest transform like the others.
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12.5pMol primer [1.25uL] of 10uM stock – be sure to use only one primer each!
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5x Buffer [5 uL]
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dNTPs (2.5mM each) [2 uL]
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Herculase II [0.5 uL]
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H2O → 25uL
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Control reaction – this is your background! Add 50uL master mix (without primers) to PCR tube and put it on the cycler, identical to the other tubes. DpnI digest transform like the others.
+
  
−
PCR Protocol:
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'''PCR Protocol''':
  
 
Stage 1: setup 2 tubes as above – one for primer, one for its complement – run 2 or 3 cycles
 
Stage 1: setup 2 tubes as above – one for primer, one for its complement – run 2 or 3 cycles
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72C, 10min
 
72C, 10min
 
Extension time – with the new faster enzymes, 30s/kB may be enough, but have had better luck with 1min
 
Extension time – with the new faster enzymes, 30s/kB may be enough, but have had better luck with 1min
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DpnI digest – add 0.5uL DpnI (10U/uL) to each 50uL reaction, vortex, incubate at 37°C for 1-3hrs (longer is better if time permits). Be sure to DpnI digest the control and transform and plate it equally. If you think the reaction is a hard one, you can extend the Dpn digestion to reduce background further (or add more enzyme).
 
  
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Stage 1:
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'''Stage 1''':
  
 
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Stage 2:
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'''Stage 2''':
  
 
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Agarose Gel – WASTE OF TIME! This technique often fails to produce enough DNA to see on a gel and still works. So, basically, this step tells you nothing, since you are going to do the transformation whether or not the gel shows you something. I would do a positive control on the transformation long before I run a gel here.
+
'''DpnI digest''' – add 0.5uL DpnI (10U/uL) to each 50uL reaction, vortex, incubate at 37°C for 1-3hrs (longer is better if time permits). Be sure to DpnI digest the control and transform and plate it equally. If you think the reaction is a hard one, you can extend the Dpn digestion to reduce background further (or add more enzyme).
  
−
PCR Cleanup – Herculase II reaction mix is incompatible with our standard competent cells (made using Mn/Ca), reducing the efficiency of transformation by at least 3 logs (perhaps due to detergent in the buffer). A quick PCR cleanup solves this problem.
+
'''Agarose Gel''' – WASTE OF TIME! This technique often fails to produce enough DNA to see on a gel and still works. So, basically, this step tells you nothing, since you are going to do the transformation whether or not the gel shows you something. I would do a positive control on the transformation long before I run a gel here.
 +
 
 +
'''PCR Cleanup''' – Herculase II reaction mix is incompatible with our standard competent cells (made using Mn/Ca), reducing the efficiency of transformation by at least 3 logs (perhaps due to detergent in the buffer). A quick PCR cleanup solves this problem.
  
 
Transform 5uL into a 50uL aliquot of competent cells. We make our own competent cells; for very difficult reactions, we have had success with supercompetent cells, but I haven’t used these since switching to PFU-Ultra. After heat shock, I grow the cells for 1hr in 500uL SOC and plate both 50uL and 450uL. Sometimes the 450uL plate is a lawn; sometimes there are only 10-50 colonies.
 
Transform 5uL into a 50uL aliquot of competent cells. We make our own competent cells; for very difficult reactions, we have had success with supercompetent cells, but I haven’t used these since switching to PFU-Ultra. After heat shock, I grow the cells for 1hr in 500uL SOC and plate both 50uL and 450uL. Sometimes the 450uL plate is a lawn; sometimes there are only 10-50 colonies.
  
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Screening – if your control plate is clean (no colonies), I recommend sequencing (or otherwise screening – if your design allows) 3 clones. Usually at least 2 are positive, but sometimes only 1. Sometimes odd things happen, such as single nucleotide insertions or deletions, errors in the primer sequence itself, or part of the primer annealing with the wrong region of the template, but these results are rarely in all clones sequenced. If there are a lot of colonies on the control (25-50% of the experimental plates), you can sometimes get lucky. Consider longer DpnI digest or repeating.
+
'''Screening''' – if your control plate is clean (no colonies), I recommend sequencing (or otherwise screening – if your design allows) 3 clones. Usually at least 2 are positive, but sometimes only 1. Sometimes odd things happen, such as single nucleotide insertions or deletions, errors in the primer sequence itself, or part of the primer annealing with the wrong region of the template, but these results are rarely in all clones sequenced. If there are a lot of colonies on the control (25-50% of the experimental plates), you can sometimes get lucky. Consider longer DpnI digest or repeating.
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+
−
 
+
  
 
===Source===
 
===Source===

Latest revision as of 23:08, 24 September 2011

pRSBB403 HIS3 Integrating Yeast Shuttle Vector


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1008
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 899
    Illegal BglII site found at 959
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1748
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3428
    Illegal SapI site found at 2345


Design Notes

The starting vector was pRS403 created by R.S. Sikorski and T.W. Christianson, et al. QuickChange PCR was used to remove restriction enzyme sites common to the multiple cloning site, in this case PstI. A further QuickChange PCR was performed to remove the existing multiple cloning site and to insert the BioBrick multiple cloning site. The QuickChange protocol is as follows:

Based on two stage protocol from - W. Wang, B. A. Malcolm, Biotechniques 26, 680 (Apr, 1999). This two-stage protocol allows formation of some of a “hybrid” between WT and mutant, reducing competition between primer and its GC

Design primer to span region to be changed. Depending on the degree of change (single point mutation vs large deletion/insertion) I usually have 15-40nt on either side with perfect match. Order primer and its reverse complement.

For each primer, setup a 25uL reaction. Make a master mix containing everything but primers and template, aliquot, and then add primers. We used Herculase II DNA Polymerase from Agilent Technologies.

Reagents Volume (uL)
Herculase 5X Buffer 5
2.5mM dNTP 2
Template DNA 1 ul from miniprep
10uM Forward Primer 1.25
10uM Reverse Primer 1.25
Herculase II Enzyme 0.5
Sterile Water Till Total
Total 25

Control reaction – this is your background! Add 50uL master mix (without primers) to PCR tube and put it on the cycler, identical to the other tubes. DpnI digest transform like the others.

PCR Protocol:

Stage 1: setup 2 tubes as above – one for primer, one for its complement – run 2 or 3 cycles Stage 2: combine the two reactions into one, mix well, run for 18 more cycles 95C, 5min [95C, 30sec / 55C, 30sec / 72C (1min/kB total vector+insert)] – 2-3x in stage 1, 18x in stage 2 72C, 10min Extension time – with the new faster enzymes, 30s/kB may be enough, but have had better luck with 1min

Stage 1:

Temperature (C) Time Cycles
95 5 mins 1
95 30 secs 3
55 30 secs 3
72 6 mins 3
72 10 mins 1

Stage 2:

Temperature (C) Time Cycles
95 5 mins 1
95 30 secs 18
55 30 secs 18
72 6 mins 18
72 10 mins 1

DpnI digest – add 0.5uL DpnI (10U/uL) to each 50uL reaction, vortex, incubate at 37°C for 1-3hrs (longer is better if time permits). Be sure to DpnI digest the control and transform and plate it equally. If you think the reaction is a hard one, you can extend the Dpn digestion to reduce background further (or add more enzyme).

Agarose Gel – WASTE OF TIME! This technique often fails to produce enough DNA to see on a gel and still works. So, basically, this step tells you nothing, since you are going to do the transformation whether or not the gel shows you something. I would do a positive control on the transformation long before I run a gel here.

PCR Cleanup – Herculase II reaction mix is incompatible with our standard competent cells (made using Mn/Ca), reducing the efficiency of transformation by at least 3 logs (perhaps due to detergent in the buffer). A quick PCR cleanup solves this problem.

Transform 5uL into a 50uL aliquot of competent cells. We make our own competent cells; for very difficult reactions, we have had success with supercompetent cells, but I haven’t used these since switching to PFU-Ultra. After heat shock, I grow the cells for 1hr in 500uL SOC and plate both 50uL and 450uL. Sometimes the 450uL plate is a lawn; sometimes there are only 10-50 colonies.

Screening – if your control plate is clean (no colonies), I recommend sequencing (or otherwise screening – if your design allows) 3 clones. Usually at least 2 are positive, but sometimes only 1. Sometimes odd things happen, such as single nucleotide insertions or deletions, errors in the primer sequence itself, or part of the primer annealing with the wrong region of the template, but these results are rarely in all clones sequenced. If there are a lot of colonies on the control (25-50% of the experimental plates), you can sometimes get lucky. Consider longer DpnI digest or repeating.

Source

pRS 403 by R.S. Sikorski and T.W. Christianson, et. al.

References