IGEM:Caltech/2007/Protocols/Recombineering Protocol

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 10: Line 10:
|style="background:#ffffff"|
|style="background:#ffffff"|
-
This protocol is adopted almost verbatim from the Oppenheim <i>et al</i>, 2004.
+
This protocol is adopted almost verbatim from Oppenheim <i>et al</i>, 2004.
<b>Recombineering</b>. The strains used for recombineering carry a defective E prophage containing
<b>Recombineering</b>. The strains used for recombineering carry a defective E prophage containing
Line 42: Line 42:
the cells transferred to chilled 35-ml centrifuge tubes and
the cells transferred to chilled 35-ml centrifuge tubes and
harvested by centrifugation at approximately 6500g for 7
harvested by centrifugation at approximately 6500g for 7
-
min. The cells are washed once with 30-ml ice-cold sterile
+
min.
 +
 
 +
6. The cells are then washed once with 30-ml ice-cold sterile
water; the pellet is quickly resuspended in 1-ml ice-cold
water; the pellet is quickly resuspended in 1-ml ice-cold
sterile water and pelleted briefly (30 s) in a refrigerated
sterile water and pelleted briefly (30 s) in a refrigerated
-
microfuge. The pellet is resuspended in 200-\mu l cold sterile
+
microfuge.
 +
 
 +
7. The pellet is resuspended in 200 microlitres cold sterile
water and 50–100 Al aliquots are used for electroporation
water and 50–100 Al aliquots are used for electroporation
with 100–150 ng PCR product or 10–100 ng oligonucleotide.
with 100–150 ng PCR product or 10–100 ng oligonucleotide.
-
We use a BioRad E. coli Gene Pulser set at 1.8 mVand
+
(Oppenheim <i>et al</i> use a BioRad E. coli Gene Pulser set at 1.8 mV with
-
0.1-cm cuvettes. Electroporated cells are diluted into 5 ml
+
0.1-cm cuvettes.)
-
39jC LB medium and incubated to allow completion of the
+
 
-
lytic cycle. The resulting phage lysate is diluted and titered on
+
8. Electroporated cells are diluted into 5 ml
-
appropriate bacteria to obtain single plaques (for more details,
+
39C LB medium and incubated to allow completion of the
-
see Thomason et al., 2003).
+
lytic cycle.
-
Oligonucleotides. The oligonucleotides were purchased
+
 
-
from Invitrogen without additional purification. The purified
+
9. The resulting phage lysate is diluted and titered on
-
oligonuclotide was subjected to electrophoresis in a 15%
+
appropriate bacteria to obtain single plaques. To screen for phages containing desired amber mutations
-
PAGE-Urea gel, excised from the gel without direct UV
+
in <i>N</i> and <i>Q</i>, the lysate can be titered in a double layer assay with amber suppressing and non-suppressing bacteria
-
irradiation and eluted using the Elutrap electro-separation
+
(see Protocols).
-
system (Schleicher and Schuell). The size-purified oligonucleotide
+
 
-
was then precipitated with isopropanol, washed with
+
 
-
ethanol, dried, and stored at �20jC.
+
 
 +
<b>Oligonucleotide Design</b>.  
 +
We purchased our oligonucleotides from IDT with PAGE purification. The oligos were homologous to the <i>N</i> and <i>Q</i> gene regions to be mutagenized, and contained a single point mutation moving a tyrosine residue to an amber stop codon.  
 +
 
|}
|}
</div>
</div>

Revision as of 15:07, 26 October 2007


iGEM 2007

Home        People        Project        Protocols        Notes        Changes       


This protocol is adopted almost verbatim from Oppenheim et al, 2004.

Recombineering. The strains used for recombineering carry a defective E prophage containing the pL operon under control of the temperature-sensitive repressor cI857.

1. The strain of choice is grown in a shaking water bath at 32C in LB with 0.4% maltose to mid-exponential phase, A600 0.4–0.6 (30 ml is adequate for several recombineering reactions).

2. The culture is harvested by centrifugation and resuspended in 1 ml TM (10mM Tris base, 10mM MgSO4, pH 7.4). The phage to be engineered is added at a multiplicity of infection of 1–3 phages/cell (we assume cell density of approximately 10^8/ml before concentration) and allowed to adsorb at room temperature for 15 min (this step would need modification for other phages, i.e., adsorption on ice).

3. Meanwhile, two flasks with 5-ml broth are pre warmed to 32C and 42C in separate shaking water baths.

4. The infected culture is divided and half inoculated into each flask; the cultures are incubated an additional 15 min. The 42C heat pulse induces prophage functions; the 32C uninduced culture is a control.

5. After induction, the flasks are well chilled in an ice water bath and the cells transferred to chilled 35-ml centrifuge tubes and harvested by centrifugation at approximately 6500g for 7 min.

6. The cells are then washed once with 30-ml ice-cold sterile water; the pellet is quickly resuspended in 1-ml ice-cold sterile water and pelleted briefly (30 s) in a refrigerated microfuge.

7. The pellet is resuspended in 200 microlitres cold sterile water and 50–100 Al aliquots are used for electroporation with 100–150 ng PCR product or 10–100 ng oligonucleotide. (Oppenheim et al use a BioRad E. coli Gene Pulser set at 1.8 mV with 0.1-cm cuvettes.)

8. Electroporated cells are diluted into 5 ml 39C LB medium and incubated to allow completion of the lytic cycle.

9. The resulting phage lysate is diluted and titered on appropriate bacteria to obtain single plaques. To screen for phages containing desired amber mutations in N and Q, the lysate can be titered in a double layer assay with amber suppressing and non-suppressing bacteria (see Protocols).


Oligonucleotide Design. We purchased our oligonucleotides from IDT with PAGE purification. The oligos were homologous to the N and Q gene regions to be mutagenized, and contained a single point mutation moving a tyrosine residue to an amber stop codon.


Personal tools