EPF-Lausanne/29 September 2008

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(Difference between revisions)
Line 28: Line 28:
#*#72°C, 5 minutes
#*#72°C, 5 minutes
#*#4°C, inf.
#*#4°C, inf.
-
#A second set of primers adds RBS (B0034) and Terminator (B0015) together with the prefix and suffix.
+
#A second set of primers adds RBS (B0034) and Terminator (B0015) together with the prefix and suffix by annealing to the "barcode" added on step 1.
#*Reaction mix:
#*Reaction mix:
-
#**We do 2 different reactions and each one to a final volume of both 25 μl and 50 μl
+
#**We do 2 different reactions one to a final volume of 25μl and one 50μl
-
#*#Same mix is used as in the first step, using the product as template.
+
#**In both cases the primers are BB5 (FW) and p230 (RV)
-
#*#Here the primer volumes are 5 μl each, otherwise same as step 1.
+
#**Same mix is used as in the first step, using the product as template.
 +
#*PCR protocol:
 +
#**Same protocol as step 1 but with annealing temperature of 60°C and only 10 cycles.
 +
#The third step just amplifies the template with primers BB3 and BB5 (that anneal to prefix and suffix).
 +
#*Reaction mix:
 +
#**25 final volume, 0.5 μl of each primer, rest as before.
 +
#*PCR protocol:
 +
#**Same protocol as step 1 but with 35 cycles.
 +
The advantage of the 2-step PCR is that we only have to order the p230 primer once, at it is longer than 100bp and thus expensive. We can add Terminator and Suffix to any part by designing just a short annealing sequence with the "barcode" as 5' overhang.
 +
 
 +
Results:
 +
 
 +
--> image of gel page 105.
 +
 
 +
The "best" band is on lane 3 (step 1 50μl, step 3 2μl). We digest it with X, P, then use PCR purification kit.
 +
 
 +
Simultaneously we digest R0071 with X, P. We want to cut out the part and use the vector to put our PCR'ed part in.
-
--> unfinished
+
We ligate, transform and plate.

Revision as of 14:35, 27 October 2008

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Cloning

2-step PCR of LuxI

The goal is to obtain the following part: BBprefix-RBS-LuxI-Terminator-BBsuffix

This is done by 2-step PCR:

  1. A first set of primers adds a "barcode"-sequence to the LuxI which is present on a the plasmid pSND (from R.Weiss).
    • Reaction mix:
      • 2.5 μl Buffer 10X
      • 0.75 μl MgCl2
      • 0.5 μl dNTP
      • 0.5 μl pSND1 (template)
      • 0.5 μl p151 (FW primer)
      • 0.5 μl p131 (RV primer)
      • 0.1 μl TAQ
      • 14.65 μl Water
    • PCR protocol:
      1. 94°C, 4 minutes
      2. 94°C, 30 seconds
      3. 55°C, 1 minute
      4. 72°C, 2 minutes
      5. cycle 2-4 30x
      6. 72°C, 5 minutes
      7. 4°C, inf.
  2. A second set of primers adds RBS (B0034) and Terminator (B0015) together with the prefix and suffix by annealing to the "barcode" added on step 1.
    • Reaction mix:
      • We do 2 different reactions one to a final volume of 25μl and one 50μl
      • In both cases the primers are BB5 (FW) and p230 (RV)
      • Same mix is used as in the first step, using the product as template.
    • PCR protocol:
      • Same protocol as step 1 but with annealing temperature of 60°C and only 10 cycles.
  3. The third step just amplifies the template with primers BB3 and BB5 (that anneal to prefix and suffix).
    • Reaction mix:
      • 25 final volume, 0.5 μl of each primer, rest as before.
    • PCR protocol:
      • Same protocol as step 1 but with 35 cycles.

The advantage of the 2-step PCR is that we only have to order the p230 primer once, at it is longer than 100bp and thus expensive. We can add Terminator and Suffix to any part by designing just a short annealing sequence with the "barcode" as 5' overhang.

Results:

--> image of gel page 105.

The "best" band is on lane 3 (step 1 50μl, step 3 2μl). We digest it with X, P, then use PCR purification kit.

Simultaneously we digest R0071 with X, P. We want to cut out the part and use the vector to put our PCR'ed part in.

We ligate, transform and plate.

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