User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/10/08

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October 8th, 2010

1. Make the following restrictions

  • pSB3K3 + J23101 SpeI-PstI restriction
  • pSB4A5 + MinBP SpeI-PstI restriction
  • SpeI-PstI double restriction methods:
DNA -> 5 ul
Buffer 2 -> 2 ul (10% of total volume)
BSA -> 1 ul
SpeI -> 1 ul
PstI -> 1 ul
HPLC -> 10 ul (to complete total volume of 20ul)
Incubate at 37º C for 4 hrs.


2. Make PCR to test the construction:

  • pSB1A2 + J23101 + ΔRBS + GFP E004 (Tubes 1-2)
  • Positive control pSB1C3 (Tube 3)
  • DNA used as template: 2ul of 1:4 dilution of extracted plasmid
  • Primers used were Preffix FWD and Suffix REV, reactives needed for one reactions are as follows:
  • PCR with Taq DNA polymerase
Reactive (ul x sample)
Taq Polymerase -> 1
Taq Reaction Buffer 10X -> 5
MgCl 50mM (can be used up to 3ul) -> 2.5
dNTP’s 0.4ug/ul -> 2.5
Primer Forward (can be used up to 3ul) -> 2.5
Primer Reverse (can be used up to 3ul) -> 2.5
HPLC -> 32
DNA -> 2
Total volume -> 50
  • Thermocycler program:
1. 95ºC 5 min
2. 35 cycles
-95ºC 45 seg
-55ºC 45 seg
-72ºC 1:10 min
3. 72ºC 10 min
4. Hold 4ºC


3. Inactivate restriction enzyme for 20 min at 80ºC.


4. Run gel tp verify PCR and restrictions.

Lanes: 1,9) Green ladder; 2) pSB1A2 + J23101 + ΔRBS + GFP E004 (Tube 1); 3) pSB1A2 + J23101 + ΔRBS + GFP E004 (Tube 2); 4) pSB1C3 (Preff FWD-Suff REV); 5) pSB3K3 + J23101 SpeI-PstI restriction; 6) pSB4A5 + MinBP SpeI-PstI restriction; 7) pSB1C3 EcoRI-PstI restriction; 8) ΔRBS + GFP E004 XbaI-PstI restriction.



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