From 2007.igem.org

Contruction of pET3a Derivatives Containing bchD and bchI

1. Amplify Rhodobacter's gene and introduce restriction sites by PCR (Using Phusion Polymerase), which allow cloning PCR fragments into pET3a

	bchD	bchI
length of gene fragment	1695 b.p.	1005 b.p.
Restriction sites introduced when amplified by PCR	NdeI, BamHI	NdeI, BglI

Rhodobacter Genomic DNA      1 ul
GC Buffer                   10 ul
dNTP                         1 ul
Primer Mix                   5 ul
Water                     27.5 ul
DMSO                       5.0 ul
Phusion Polymerase         0.5 ul

bchD
1.Initial Denaturation	2.Denaturation	3.Annealing	4.Extension	5.Repeat step 2 to 4 for 30 times	6.Final Extension
98 °C	98 °C	52 °C	72 °C		72 °C	4 °C
30 sec	8 sec	30 sec	60 sec		10 min	forever

bchI
1.Initial Denaturation	2.Denaturation	3.Annealing	4.Extension	5.Repeat step 2 to 4 for 30 times	6.Final Extension
98 °C	98 °C	62 °C	72 °C		72 °C	4 °C
30 sec	8 sec	30 sec	32 sec		10 min	forever

2. Do DNA Gel Electrophoresis to confirm that the length of the PCR fragments are the same as the corresponding target gene fragments.

3. Remove any leftover PCR enzyme in the samples by PCR Clean Up/Purification.

4. Digestion for PCR Products using specific restriction enzymes

bchD	NdeI, BamHI
bchI	NdeI, BglI

4. Digested bchD and bchI are subjected to Gel Extraction to ensure that only pure, digested DNA is obtained before doing ligation.

5. Ligate digested and purified PCR fragments with digested pET3a by Ligation yielding plasmids of pETBCHD and pETBCHI.

6. Use small portion of the plasmids to do Analytic Digestion.

7. Do KCM Competent Cell Transformation to subclone plasmids into DH10B competent cells, which have been done by KCM Competent Cell Production.

8. Pick colonies and innoculate in 2ml LB + 2ul Carbencilin in 37°C shaker overnight.

9. Miniprep

10. Analytic Digestion.

11. Digestion for Miniprepped DNA

12. Do KCM Competent Cell Transformation to subclone plasmids into BL21, which have been done by KCM Competent Cell Production.

Protein Expression

Protein Analysis