Difference between revisions of "Part:BBa K332011:Experience"

(→‎Applications of BBa_K332011)
(→‎Applications of BBa_K332011)
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4. Find the best PCR condition by gradient PCR.  
 
4. Find the best PCR condition by gradient PCR.  
 
   Anneling temperature: 51°C±10°C
 
   Anneling temperature: 51°C±10°C
 +
 +
  [[Image:gradient PCR.jpg]]
  
 
5. PCR by B-taq plus on the best condition
 
5. PCR by B-taq plus on the best condition
Line 45: Line 47:
 
   ddH2O                             34.0   
 
   ddH2O                             34.0   
 
   Total             50 μl
 
   Total             50 μl
 +
   
 +
  94°C  5  min
 +
  94°C  30  sec
 +
  53°C  30  sec
 +
  72°C  2.5 min
 +
  72°C  10  min
 +
  cycles:25
  
 
6. Digestion to confirm the cry11Aa fragment and enzyme sites.
 
6. Digestion to confirm the cry11Aa fragment and enzyme sites.
 +
  [[Image:cry digestion.jpg]]
  
−
7. TA clone
+
7. TA cloning
  
 
8. DNA sequencing
 
8. DNA sequencing
 +
  [[Image:cry11Aa sequencing.jpg]]
  
 
(III) PCR mutagenesis at two enzyme sites --- EcoR1 and Spe1
 
(III) PCR mutagenesis at two enzyme sites --- EcoR1 and Spe1
Line 64: Line 75:
  
 
1. Design primers by Assembly standard 10.
 
1. Design primers by Assembly standard 10.
−
   prefix: GTTTCTTC GAATTC GCGGCCGC T TCTAG ATGGAAGATAGTTCTTTAGATACT
+
   prefix: GTTTCTTC GAATTC GCGGCCGC T TCTAG ATGGAAGATAGTTCTTTAGATACTTTAAG
−
   suffix: GTTTCTTC CTGCAG CGGCCGC  T ACTAGT ACTACTTTAGTAACGGATT        
+
   suffix: GTTTCTTC CTGCAG CGGCCGC  T ACTAGT ACTACTTTAGTAACGGATTAATTTGC        
  
 
2. PCR condition
 
2. PCR condition
−
     53
+
 
 +
     95°C  30  sec
 +
    95°C  30  sec
 +
    55°C  1  min
 +
    72°C  2.5 min
 +
    72°C  10  min
 +
    cycles:15
 +
 
 
3. Ligation to backbones(Psb1C3).
 
3. Ligation to backbones(Psb1C3).
  
 
(V)  Transform into E.coli
 
(V)  Transform into E.coli
  
−
1. Thaw competent cells and BBa_K332012 plasmid on ice.
+
1. Thaw competent cells and BBa_K332011 plasmid on ice.
  
 
2. Add 2ul plasmid to competent cell and place in ice for 5 minutes.
 
2. Add 2ul plasmid to competent cell and place in ice for 5 minutes.
Line 83: Line 101:
 
5. Incubate the cultures at 37°C overnight.
 
5. Incubate the cultures at 37°C overnight.
  
−
colony PCR EXSP
+
6. colony PCR to confirm the fragment size by (prefix & suffix primers)
 +
 
 +
7. DNA sequencing:
 +
  [[Image:cry11Aa part sequencing.JPG]]
  
 
(VI)  Culture with mosquito larvae and observe
 
(VI)  Culture with mosquito larvae and observe

Revision as of 01:44, 29 October 2010

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K332011

(I) Culture Bti.

Bacillus thuringiensis subsp. Israelensis (BCRC15860)

1. Prepare agar plate for Bti. 

  Beef extract  3.0g
  Peptone       5.0g
  Agar          15.0g
  ddH2O         1 L
  • adjust PH to 7.0
  • No antibiotic
  • Be aware of contamination

2. Plate Bti. on the medium and incubate the cultures at 30°C overnight.

(II) Clone cry11Aa from Bti. into TA vector

1. Extract genomic DNA of Bti. by liquid nitrogen.

2. Add some ddH2O to dilute the DNA.

3. Design primers 

  cry forward:  ATTCAATAAAAGGTGGAATGAATTATGGA       Tm: 53°C
  cry reverse:  GTGCTAACATGACTTCTACTTTAGT           Tm: 52.8°C

4. Find the best PCR condition by gradient PCR. 

  Anneling temperature: 51°C±10°C

  Gradient PCR.jpg

5. PCR by B-taq plus on the best condition 

  Template DNA（10ng/μl）		    2.0
  B-taq buffer			            5.0
  dNTP（2mM）			            5.0
  forward primer（10μM）		            1.5
  reverse primer（10μM）		            1.5
  B-taq plus DNA polymerase（2Kb）           1.0
  ddH2O		                            34.0  
  Total				            50 μl
   
  94°C   5  min
  94°C  30  sec
  53°C  30  sec
  72°C  2.5 min
  72°C  10  min
  cycles:25

6. Digestion to confirm the cry11Aa fragment and enzyme sites. 

  Cry digestion.jpg

7. TA cloning

8. DNA sequencing 

  Cry11Aa sequencing.jpg

(III) PCR mutagenesis at two enzyme sites --- EcoR1 and Spe1

1. Design primers by primerX. 

  EcoR1-f: CGG GTA CAA TCT CAG AAC TCG GGA AAT AAT AGA A
  EcoR1-R: TTC TAT TAT TTC CCG AGT TCT GAG ATT GTA CCC G
  Spe1-f:  ATA ATG AAT GGG GAG GAC TGG TTT ATA AGT TAT TAA TGG G
  Spe1-R:  CTT CCC CCA TTA ATA ACT TAT AAA CTA GTC CTC CCC ATT CAT

2. Digestion to confirm the fragments

(IV) PCR construction of Biobrick parts

1. Design primers by Assembly standard 10. 

  prefix: GTTTCTTC GAATTC GCGGCCGC T TCTAG ATGGAAGATAGTTCTTTAGATACTTTAAG
  suffix: GTTTCTTC CTGCAG CGGCCGC  T ACTAGT ACTACTTTAGTAACGGATTAATTTGC

2. PCR condition 

   95°C  30  sec
   95°C  30  sec
   55°C   1  min
   72°C  2.5 min
   72°C  10  min
   cycles:15

3. Ligation to backbones(Psb1C3).

(V) Transform into E.coli

1. Thaw competent cells and BBa_K332011 plasmid on ice.

2. Add 2ul plasmid to competent cell and place in ice for 5 minutes.

3. Put the transformed cells into 42℃ water bath for 45 seconds.

4. Plate the cells on the appropriate media and antibiotic, such as agar plates with 25 µg/ml kanamycin.

5. Incubate the cultures at 37°C overnight.

6. colony PCR to confirm the fragment size by (prefix & suffix primers)

7. DNA sequencing: 

  Cry11Aa part sequencing.JPG

(VI) Culture with mosquito larvae and observe

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