User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/09/14

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September 14th, 2010

1. Gel to verify PCR made on September 13th.

Lanes: 1) Green ladder; 2-10) Paz’s samples; 11) BBa_I20260 Suffix FWD-J23101 REV reaction 1; 12) BBa_I20260 Suffix FWD-J23101 REV reaction 2; 13) BBa_I20260 RBS GFP FWD-Suffix REV reaction 1; 14) BBa_I20260 RBS GFP FWD-Suffix REV reaction 2; 15) Green ladder.

  • There are inespecific bands in the gel besides the expected products, I’ll repeat the PCR to try to eliminate these inespecific bands.


2. Repeat PCR to amplify BBa_I20260 (pSB3K3 + J23101 promoter + GFP E0040), one reaction will amplify the plasmid including the J23101 promoter, another reaction will amplify only the GFP E0040 and a third reaction will amplify pSB3K3 plasmid and insert into this plasmid our minimum blue promoter.

  • PCR will be done with Platinum Taq Polymerase.
  • I will make 2 reactions with primers Suffix FWD-J23101 REV (Tubes 1-2).
  • PCR product is expected to be 3670 bp (35 promoter + 2729 plasmid pSB3K3).
  • To amplify the GFP E0040: RBS GFP FWD-Suffix REV (Tubes 3-4) GFP length is 720 bp.
  • PCR to insert minimum blue promoter into pSB3K3 will be done with minBP REV-Suffix FWD primers.
  • Template for the three reactions is BBa_I20260 plasmid diluted 1:4
PCR with Platinum Taq DNA polymerase Volume (ul)
10X PCR Buffer minus M -> 5
10mM dNTP mixture -> 1
50mM MgCl2 -> 1.5
Primer mix (10uM each) -> 1
Platinum Taq DNA Pol -> 0.4
Template DNA -> 2
HPLC -> 35.1
Total volume -> 50
  • If primers are separated and in concentration 5uM, use 1ul of each one.
  • Thermocycler program:
1. 95ºC 5 min
2. 35 cycles
  • 95ºC 45 seg
  • 55ºC 45 seg
  • 72ºC 4 min
3. 72ºC 10 min
4. Hold 4ºC


3. Run gel to verify PCR.

Lanes: 1) Green ladder; 2) BBa_I20260 Suffix FWD-J23101 REV reaction 1 (tube 1); 3) BBa_I20260 Suffix FWD-J23101 REV reaction 2 (tube 2); 4) BBa_I20260 RBS GFP FWD-Suffix REV reaction 1 (tube 3); 5) BBa_I20260 RBS GFP FWD-Suffix REV reaction 2 (tube 4); 6) BBa_I20260 MinBP REV-Suffix FWD primers (tube 5); 7) BBa_I20260 MinBP REV-Suffix FWD primers (tube 6).


4. Purify PCR products from BBa_I20260 Suffix FWD-J23101 REV (reactions 1 and 2) and BBa_I20260 RBS GFP FWD-Suffix REV (reactions 1 and 2) using the High Pure PCR Product Purification Kit Roche.


5. Make restrictions to pure PCR products, plasmid pSB3K3 with promoter J23101 will be restricted with SpeI-PstI and GFP E0040 with XbaI-PstI.

  • SpeI-PstI double restriction methods:
DNA -> 15 ul
Buffer 2 -> 4 ul (10% of total volume)
BSA -> 1 ul
SpeI -> 2 ul
PstI -> 2 ul
HPLC -> 16 ul (to complete total volume of 20ul)
  • XbaI-PstI double restriction methods:
DNA -> 15 ul
Buffer 2 -> 4 ul (10% of total volume)
BSA -> 1 ul
XbaI -> 2 ul
PstI -> 2 ul
HPLC -> 16 ul (to complete total volume of 20ul)
  • Incubate at 37ºC overnight.


6. Repeat PCR to amplify BBa_I20260 (pSB3K3 + J23101 promoter + GFP E0040) and insert into this plasmid our minimum blue promoter.

  • PCR will be done with Platinum Taq Polymerase.
  • I will make 2 reactions with primers Min-BP REV-Suffix FWD (Tubes 1-2).
  • PCR product is expected to be 3670 bp (35 promoter + 2729 plasmid pSB3K3).
  • Template for the reaction is BBa_I20260 plasmid diluted 1:4
PCR with Platinum Taq DNA polymerase Volume (ul)
10X PCR Buffer minus M -> 5
10mM dNTP mixture -> 1
50mM MgCl2 -> 1.5
Primer mix (10uM each) -> 1
Platinum Taq DNA Pol -> 0.4
Template DNA -> 2
HPLC -> 35.1
Total volume -> 50
  • If primers are separated and in concentration 5uM, use 1ul of each one.
  • Thermocycler program:
1. 95ºC 5 min
2. 35 cycles
  • 95ºC 45 seg
  • 55ºC 45 seg
  • 72ºC 4 min
3. 72ºC 10 min
4. Hold 4ºC



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