Template:SBB-Protocols Enz6

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In this protocol, you are trying to 'map' your plasmid to determine whether it is likely to be the desired sequence.  There are two pieces of information you need to consider before setting up the digest:
 +
 +
*  How much DNA should I use?
 +
*  What restriction enzymes should I use?
 +
 +
The choice of how much of your miniprepped plasmid to add depends on the concentration of DNA in the sample, which primarily reflects the copy number of the plasmid in the cell, which is primarily determined by what origin of replication is on the plasmid.  However, subtleties about how you do the miniprep procedure, what else is on the plasmid, and any stress/load present in the bacterium can affect the plasmid yield.  To a first approximation, you can make this decision based on the origin of replication:
 +
 +
  colE1 (pUC plasmids, such as pBca9145, pBca9523, pBca9525 vectors)  :  use 1uL as it is very high copy
 +
  colE1+ROP  (pBR322-derived plasmids that retain ROP)  :  use 2uL as it's a little lower when ROP is present
 +
  p15A  (such as pBgl0001 and pAC vectors)    :    use 4uL as it is medium copy
 +
  pSC101 :  use 6 uL as it's lower copy
 +
  F plasmid (such as BACs)  :  use 8uL since these are very low copy
 +
 +
Just use that volume of DNA, and adjust the amount of water so that things add up to 10uL
 +
 +
The choice of what restriction enzymes to use involves using ApE (or an equivalent tool that predicts the fragmentation pattern of a restriction digest).  Your primary goal is to distinguish your product from the plasmid that you inserted your part into.  Often that means, say, distinguishing pBca9145-Bca1144#5 from your product pBca9145-sbb1222.  The reason for this is that primarily what bleeds through onto the plate is this parent plasmid, or mutants thereof.  So, you want to find one or two enzymes that gives a clearly-different restriction pattern in your product from that of the parent vector.
 +
 +
Scenarios:
 +
 +
*  A good place to start is considering using combinations of the BioBrick format enzymes (for BglBricks, that means EcoRI, BamHI, BglII, XhoI) or the enzymes you used during your cloning.  If the part that you inserted is significantly larger or smaller than the one in the parent vector, this is a very good strategy as you'll get 2 bands for both parent and product, and you can tell by the size which is which.
 +
*  Sometimes your part is too similar in size to the parent part.  Under these circumstances, look for a restriction site that is in the product part but absent in the parent part.  Use that enzyme in combination with one of the BioBrick enzymes.
 +
*  Sometimes your part is too short to see well on the gel, even though it is readily distinguishable from the parent part.  Here, you should consider using other enzymes in the backbone of both plasmids in conjunction with a BioBrick enzyme to get a unique pattern.  AlwNI is often useful in this regard.
 +
*  'fingerprinting' the plasmids with DpnI often will disambiguate hard-to-distinguish cases.  Here, you use an enzyme that cuts many times in the plasmids, and the fragmentation pattern will often give a different spectrum in the parent and product.
 +
Set up the following 10uL reaction in a PCR tube:
Set up the following 10uL reaction in a PCR tube:
<pre>
<pre>
-
4uL ddH2O
+
7 uL ddH2O
-
4uL Miniprepped plasmid
+
1uL Miniprepped plasmid
1uL 10x NEB Buffer 2
1uL 10x NEB Buffer 2
-
1uL EcoRI
+
0.5uL EcoRI
-
1uL BamHI (for parts >250bp) or XhoI (for parts <250bp)
+
0.5uL BamHI
-
FOR Righty Methylated Parts use Eco/Xho!
+
</pre>
</pre>

Current revision

In this protocol, you are trying to 'map' your plasmid to determine whether it is likely to be the desired sequence. There are two pieces of information you need to consider before setting up the digest:

  • How much DNA should I use?
  • What restriction enzymes should I use?

The choice of how much of your miniprepped plasmid to add depends on the concentration of DNA in the sample, which primarily reflects the copy number of the plasmid in the cell, which is primarily determined by what origin of replication is on the plasmid. However, subtleties about how you do the miniprep procedure, what else is on the plasmid, and any stress/load present in the bacterium can affect the plasmid yield. To a first approximation, you can make this decision based on the origin of replication:

 colE1 (pUC plasmids, such as pBca9145, pBca9523, pBca9525 vectors)  :   use 1uL as it is very high copy
 colE1+ROP  (pBR322-derived plasmids that retain ROP)   :  use 2uL as it's a little lower when ROP is present
 p15A  (such as pBgl0001 and pAC vectors)    :    use 4uL as it is medium copy
 pSC101 :  use 6 uL as it's lower copy
 F plasmid (such as BACs)   :  use 8uL since these are very low copy

Just use that volume of DNA, and adjust the amount of water so that things add up to 10uL

The choice of what restriction enzymes to use involves using ApE (or an equivalent tool that predicts the fragmentation pattern of a restriction digest). Your primary goal is to distinguish your product from the plasmid that you inserted your part into. Often that means, say, distinguishing pBca9145-Bca1144#5 from your product pBca9145-sbb1222. The reason for this is that primarily what bleeds through onto the plate is this parent plasmid, or mutants thereof. So, you want to find one or two enzymes that gives a clearly-different restriction pattern in your product from that of the parent vector.

Scenarios:

  • A good place to start is considering using combinations of the BioBrick format enzymes (for BglBricks, that means EcoRI, BamHI, BglII, XhoI) or the enzymes you used during your cloning. If the part that you inserted is significantly larger or smaller than the one in the parent vector, this is a very good strategy as you'll get 2 bands for both parent and product, and you can tell by the size which is which.
  • Sometimes your part is too similar in size to the parent part. Under these circumstances, look for a restriction site that is in the product part but absent in the parent part. Use that enzyme in combination with one of the BioBrick enzymes.
  • Sometimes your part is too short to see well on the gel, even though it is readily distinguishable from the parent part. Here, you should consider using other enzymes in the backbone of both plasmids in conjunction with a BioBrick enzyme to get a unique pattern. AlwNI is often useful in this regard.
  • 'fingerprinting' the plasmids with DpnI often will disambiguate hard-to-distinguish cases. Here, you use an enzyme that cuts many times in the plasmids, and the fragmentation pattern will often give a different spectrum in the parent and product.

Set up the following 10uL reaction in a PCR tube:

7 uL ddH2O
1uL Miniprepped plasmid
1uL 10x NEB Buffer 2
0.5uL EcoRI
0.5uL BamHI
  • Incubate at 37 on the thermocycler for 30 minutes
  • Run an analytical gel
  • Take a picture of the gel
  • Calculate the expected fragment sizes
  • Are the calculated sizes consistent with the bands on the gel?
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