Team:Washington/Project/Tools/Mougous

From 2010.igem.org

(Difference between revisions)
(New page: Recombineering)
Line 1: Line 1:
-
Recombineering
+
=''galK'' Recombineering=
 +
 
 +
1. Design ''galK'' primers with 50 bp homology to an area flanking the desired
 +
site to be modified. The 3’ end of these primers bind to the ''galK'' cassette.
 +
For example, if you want to make a single bp substitution, your homology
 +
arms should be the 50 bp on either side of the single bp. This will result in
 +
a deletion of that basepair in the first step. If you want to make a small or a
 +
large deletion, design the ''galK'' primers so the deletion is made already in
 +
the first step. The primers should look as follows:
 +
 
 +
Forward:
 +
5’------------50bp_homology-------CCTGTTGACAATTAATCATCGGCA-3’
 +
Reverse:
 +
5’----50bp_homology_compl._strand----TCAGCACTGTCCTGCTCCTT-3’
 +
 
 +
2. Design other primers/oligos depending on what the desired modification
 +
is. If point mutation, design two 100 bp complementary oligos with the
 +
modified basepair(s) in the middle. The remaining bases on either side are
 +
homology arms. If a deletion is made with the ''galK'' cassette in the first
 +
step, design two complementary oligos to substitute for ''galK''. This will
 +
result in a “seam-less” deletion, leaving nothing but the desired change
 +
behind.
 +
 
 +
3. Choose a BAC for your purpose. If you need a 129 BAC for subsequent
 +
construction of a targeting vector, screen a 129 library like the CITB library
 +
from Invitrogen. Order the clones from Invitrogen. End sequence the
 +
positive clones to find one that contains the sequence needed. If a
 +
C57BL/6 BAC is needed, identify a BAC using a genome browser (like
 +
http://genome.UCSC.edu/), and order the BAC from Invitrogen or CHORI.
 +
Before proceeding, make sure the BAC is 100% correct; PCR analysis
 +
and restriction enzymatic analysis (fingerprinting). We have had good
 +
succes with SpeI digests – it cuts frequently enough to give many bands,
 +
and infrequently enough so that the band sizes are informative. It’s
 +
worthwhile spending some time on this BAC characterization – if you
 +
don’t, you might regret it later…
 +
 
 +
4. Transform the characterized BAC into electrocompetent SW102 cells (see
 +
recombineering protocol #1). Recover for 1 hour at 32°C, and plate on LB
 +
plates with 12.5 mg/ml chloramphenicol.
 +
 
 +
5. PCR amplify the ''galK'' cassette using the primers designed in step 1 and a
 +
proof-reading Taq-mix (we use Expand High Fidelity from Roche). Use 1-2
 +
ng template (the pgalK plasmid). 94°C 15 sec., 60°C 30 sec., 72°C 1 min.,
 +
for 30 cycles. Add 1-2 ml DpnI per 25 ml reaction, mix, and incubate at
 +
37°C for 1 hour. This step serves to remove any plasmid template;
 +
plasmid is methylated, PCR products are not. Gel-purify the DpnI-digested
 +
PCR product, preferably overnight at low voltage. From a strong PCR
 +
band, purified, and eluted in 50 ml ddH2O, we use 2.5 ml for a
 +
transformation (approx. 10-30 ng).
 +
 
 +
6. Inoculate an overnight culture of SW102 cells containing the BAC. 5 ml LB
 +
+ chloramphenicol. Incubate at 32°C.
 +
 
 +
7. Next day, turn on two shaking waterbaths: One at 32°C, the other at 42°C.
 +
Make an ice/water slurry and put a 50 ml tube of ddH2O in there to make
 +
sure it’s ice-cold (see later). Dilute 500 ml of the overnight SW102 culture
 +
containing the target BAC in 25 ml LB with chloramphenicol (12.5 mg/ml) in
 +
a 50 ml baffled conical flask and incubate at 32°C in a shaking waterbath
 +
to an OD600 of approx. 0.6 (0.55-0.6). This usually takes 3-4 hours.
 +
8. Transfer 10 ml to another baffled 50 ml conical flask and heat-shock at
 +
42°C for exactly 15 min. in a shaking waterbath. The remaining culture is
 +
left at 32°C as the un-induced control.
 +
 
 +
9. After 15 min., the two samples, induced and un-induced, are briefly cooled
 +
in an ice/waterbath slurry and then transferred to two 15 ml Falcon tubes
 +
and pelleted using 5000 RPM at 0°C for 5 min. It’s important to keep the
 +
bacteria as close to 0°C as possible in order to get good competents cells.
 +
 
 +
10. Pour off all of the supernatant and resuspend the pellet in 1 ml ice-cold
 +
ddH2O by gently swirling the tubes in the ice/waterbath slurry. No
 +
pipetting. This step may take a while. When resuspended, add another 9
 +
ml ice-cold ddH2O and pellet the samples again.
 +
 
 +
11. Repeat step 10.
 +
 
 +
12. After the second washing and centrifugation step, all supernatant must be
 +
removed by inverting the tubes on a paper towel, and the pellet
 +
(approximately 50 ml each) is kept on ice until electroporated with PCR
 +
product.
 +
 
 +
13. Transform the now electrocompetent SW102 cells. We use 25 ml cells for
 +
each electroporation in a 0.1 cm cuvette (BioRad) at 25 mF, 1.75 kV, and
 +
200 ohms. After electroporation of the PCR product, the bacteria are
 +
recovered in 1 ml LB (15 ml Falcon tube) for 1 hour in a 32°C shaking
 +
waterbath.
 +
 
 +
14. After the recovery period the bacteria are washed twice in 1xM9 salts as follows: 1 ml culture is pelleted in an eppendorf tube at
 +
13,200 RPM for 15 sec. and the supernatant removed with a pipette. The
 +
pellet is resuspended in 1 ml 1xM9 salts, and pelleted again. This washing
 +
step is repeated once more. After the second wash, the supernatant is
 +
removed and the pellet is resuspended in 1 ml 1xM9 salts before plating
 +
serial dilutions in 1xM9 (100 ml, 100 ml of a 1:10 dilution, and 100 ml 1:100)
 +
onto M63 minimal media plates with galactose, leucine,
 +
biotin, and chloramphenicol. Washing in M9 salts is necessary to remove
 +
any rich media from the bacteria prior to selection on minimal media.
 +
 
 +
15. Incubate 3 days at 32°C in a cabinet-type incubator.
 +
 
 +
16. Streak a few colonies onto MacConkey + galactose + chloramphenicol
 +
indicator plates. Streak to obtain single colonies (see appendix B). The
 +
colonies appearing after the 3 days of incubation should be Gal+, but in
 +
order to get rid of any Gal- contaminants (hitch-hikers), it is important to
 +
obtain single, bright red colonies before proceeding to the second step.
 +
Gal- colonies will be white/colorless and the Gal+ bacteria will be bright
 +
red/pink due to a pH change resulting from fermented galactose after an
 +
overnight incubation at 32°C.
 +
 
 +
17. Pick a single, bright red (Gal+) colony and inoculate a 5 ml LB +
 +
chloramphenicol overnight culture. Incubate at 32°C. There is normally no
 +
need to further characterize the clones after the first step.
 +
 
 +
18. Repeat steps 7 through 12 above to obtain electrocompetent SW102 cells
 +
(now ready for a ''galK'' <> mutation substitution). If you are going to
 +
transform a double-stranded DNA oligo, the two complementary oligos
 +
can be annealed in vitro: Mix 10 mg of each oligo in a volume of 100 ml 1x
 +
PCR buffer. Boil for 5 min. Let cool slowly to room temp (30 min.). Add 10
 +
ml 3 M NaAc and 250 ml EtOH. Precipitate, wash once in 70% EtOH, and
 +
resuspend the final, air-dried, pellet in 100 ml ddH2O (final conc. of 200
 +
ng/ml). Use 1 ml per transformation.
 +
 
 +
19. Transform the bacteria (25 ml of heat-shocked and 25 ml of uninduced
 +
control) with 200 ng double-stranded oligo, a PCR product, or anything
 +
containing a mutation and with homology to the area flanking the ''galK''
 +
cassette. Recover in 10 ml LB in a 50 ml baffled conical flask by
 +
incubating in a 32°C shaking waterbath for 4.5 hours. This long recovery
 +
period serves to obtain bacteria, by “dilution”, that only contains the
 +
desired recombined BAC, and thus have lost any BAC still containing the
 +
''galK'' cassette.
 +
 
 +
20. As in step 14, pellet 1 ml culture and wash twice in 1xM9 salts, and
 +
resuspend in 1 ml 1xM9 salts after the second wash before plating serial
 +
dilutions (100 ml, 100 ml of a 1:10 dilution, 100 ml 1:100, and 100 ml 1:1000)
 +
on M63 minimal media plates with glycerol, leucine, biotin, 2-deoxygalactose
 +
(DOG), and chloramphenicol.
 +
 
 +
21. Incubate at 32°C for three days.
 +
 
 +
22. The number of colonies may or may not be significantly different when
 +
comparing plates from uninduced and induced bacteria (range between
 +
1:1 – 1:100). In either case, you will still be able to find true recombinants
 +
with a high frequency. Analyze, say, 10-12 colonies by SpeI digestion of
 +
BAC miniprep DNA (see appendix C). Include a SpeI digest of the parent
 +
BAC as a control. Clones with a digestion pattern like the parent are likely
 +
to have undergone the desired mutation. Background clones (DOG
 +
resistant without the desired mutation) willl have obvious deletions, and
 +
should not be analyzed further. The clones with correct digestion pattern
 +
should be analyzed by PCR and sequencing of the mutated region. If the
 +
pattern is identical to the parental digestion pattern, it means that the
 +
bacteria most likely became DOG resistant due to the desired homologous
 +
recombination event.
 +
Alternatively, any deletion that contains the region
 +
with the ''galK'' cassette, but excludes the chloramphenicol region, will also
 +
be selected in the counterselection procedure. With a high frequency of
 +
homologous recombination as in he SW102 strain, the background is not
 +
likely to be a problem. In the unlikely event that too high a background is
 +
observed, try to increase the length of the homology arms, to increase the
 +
frequency of homologous recombination.

Revision as of 20:06, 11 October 2010

galK Recombineering

1. Design galK primers with 50 bp homology to an area flanking the desired site to be modified. The 3’ end of these primers bind to the galK cassette. For example, if you want to make a single bp substitution, your homology arms should be the 50 bp on either side of the single bp. This will result in a deletion of that basepair in the first step. If you want to make a small or a large deletion, design the galK primers so the deletion is made already in the first step. The primers should look as follows:

Forward: 5’------------50bp_homology-------CCTGTTGACAATTAATCATCGGCA-3’ Reverse: 5’----50bp_homology_compl._strand----TCAGCACTGTCCTGCTCCTT-3’

2. Design other primers/oligos depending on what the desired modification is. If point mutation, design two 100 bp complementary oligos with the modified basepair(s) in the middle. The remaining bases on either side are homology arms. If a deletion is made with the galK cassette in the first step, design two complementary oligos to substitute for galK. This will result in a “seam-less” deletion, leaving nothing but the desired change behind.

3. Choose a BAC for your purpose. If you need a 129 BAC for subsequent construction of a targeting vector, screen a 129 library like the CITB library from Invitrogen. Order the clones from Invitrogen. End sequence the positive clones to find one that contains the sequence needed. If a C57BL/6 BAC is needed, identify a BAC using a genome browser (like http://genome.UCSC.edu/), and order the BAC from Invitrogen or CHORI. Before proceeding, make sure the BAC is 100% correct; PCR analysis and restriction enzymatic analysis (fingerprinting). We have had good succes with SpeI digests – it cuts frequently enough to give many bands, and infrequently enough so that the band sizes are informative. It’s worthwhile spending some time on this BAC characterization – if you don’t, you might regret it later…

4. Transform the characterized BAC into electrocompetent SW102 cells (see recombineering protocol #1). Recover for 1 hour at 32°C, and plate on LB plates with 12.5 mg/ml chloramphenicol.

5. PCR amplify the galK cassette using the primers designed in step 1 and a proof-reading Taq-mix (we use Expand High Fidelity from Roche). Use 1-2 ng template (the pgalK plasmid). 94°C 15 sec., 60°C 30 sec., 72°C 1 min., for 30 cycles. Add 1-2 ml DpnI per 25 ml reaction, mix, and incubate at 37°C for 1 hour. This step serves to remove any plasmid template; plasmid is methylated, PCR products are not. Gel-purify the DpnI-digested PCR product, preferably overnight at low voltage. From a strong PCR band, purified, and eluted in 50 ml ddH2O, we use 2.5 ml for a transformation (approx. 10-30 ng).

6. Inoculate an overnight culture of SW102 cells containing the BAC. 5 ml LB + chloramphenicol. Incubate at 32°C.

7. Next day, turn on two shaking waterbaths: One at 32°C, the other at 42°C. Make an ice/water slurry and put a 50 ml tube of ddH2O in there to make sure it’s ice-cold (see later). Dilute 500 ml of the overnight SW102 culture containing the target BAC in 25 ml LB with chloramphenicol (12.5 mg/ml) in a 50 ml baffled conical flask and incubate at 32°C in a shaking waterbath to an OD600 of approx. 0.6 (0.55-0.6). This usually takes 3-4 hours. 8. Transfer 10 ml to another baffled 50 ml conical flask and heat-shock at 42°C for exactly 15 min. in a shaking waterbath. The remaining culture is left at 32°C as the un-induced control.

9. After 15 min., the two samples, induced and un-induced, are briefly cooled in an ice/waterbath slurry and then transferred to two 15 ml Falcon tubes and pelleted using 5000 RPM at 0°C for 5 min. It’s important to keep the bacteria as close to 0°C as possible in order to get good competents cells.

10. Pour off all of the supernatant and resuspend the pellet in 1 ml ice-cold ddH2O by gently swirling the tubes in the ice/waterbath slurry. No pipetting. This step may take a while. When resuspended, add another 9 ml ice-cold ddH2O and pellet the samples again.

11. Repeat step 10.

12. After the second washing and centrifugation step, all supernatant must be removed by inverting the tubes on a paper towel, and the pellet (approximately 50 ml each) is kept on ice until electroporated with PCR product.

13. Transform the now electrocompetent SW102 cells. We use 25 ml cells for each electroporation in a 0.1 cm cuvette (BioRad) at 25 mF, 1.75 kV, and 200 ohms. After electroporation of the PCR product, the bacteria are recovered in 1 ml LB (15 ml Falcon tube) for 1 hour in a 32°C shaking waterbath.

14. After the recovery period the bacteria are washed twice in 1xM9 salts as follows: 1 ml culture is pelleted in an eppendorf tube at 13,200 RPM for 15 sec. and the supernatant removed with a pipette. The pellet is resuspended in 1 ml 1xM9 salts, and pelleted again. This washing step is repeated once more. After the second wash, the supernatant is removed and the pellet is resuspended in 1 ml 1xM9 salts before plating serial dilutions in 1xM9 (100 ml, 100 ml of a 1:10 dilution, and 100 ml 1:100) onto M63 minimal media plates with galactose, leucine, biotin, and chloramphenicol. Washing in M9 salts is necessary to remove any rich media from the bacteria prior to selection on minimal media.

15. Incubate 3 days at 32°C in a cabinet-type incubator.

16. Streak a few colonies onto MacConkey + galactose + chloramphenicol indicator plates. Streak to obtain single colonies (see appendix B). The colonies appearing after the 3 days of incubation should be Gal+, but in order to get rid of any Gal- contaminants (hitch-hikers), it is important to obtain single, bright red colonies before proceeding to the second step. Gal- colonies will be white/colorless and the Gal+ bacteria will be bright red/pink due to a pH change resulting from fermented galactose after an overnight incubation at 32°C.

17. Pick a single, bright red (Gal+) colony and inoculate a 5 ml LB + chloramphenicol overnight culture. Incubate at 32°C. There is normally no need to further characterize the clones after the first step.

18. Repeat steps 7 through 12 above to obtain electrocompetent SW102 cells (now ready for a galK <> mutation substitution). If you are going to transform a double-stranded DNA oligo, the two complementary oligos can be annealed in vitro: Mix 10 mg of each oligo in a volume of 100 ml 1x PCR buffer. Boil for 5 min. Let cool slowly to room temp (30 min.). Add 10 ml 3 M NaAc and 250 ml EtOH. Precipitate, wash once in 70% EtOH, and resuspend the final, air-dried, pellet in 100 ml ddH2O (final conc. of 200 ng/ml). Use 1 ml per transformation.

19. Transform the bacteria (25 ml of heat-shocked and 25 ml of uninduced control) with 200 ng double-stranded oligo, a PCR product, or anything containing a mutation and with homology to the area flanking the galK cassette. Recover in 10 ml LB in a 50 ml baffled conical flask by incubating in a 32°C shaking waterbath for 4.5 hours. This long recovery period serves to obtain bacteria, by “dilution”, that only contains the desired recombined BAC, and thus have lost any BAC still containing the galK cassette.

20. As in step 14, pellet 1 ml culture and wash twice in 1xM9 salts, and resuspend in 1 ml 1xM9 salts after the second wash before plating serial dilutions (100 ml, 100 ml of a 1:10 dilution, 100 ml 1:100, and 100 ml 1:1000) on M63 minimal media plates with glycerol, leucine, biotin, 2-deoxygalactose (DOG), and chloramphenicol.

21. Incubate at 32°C for three days.

22. The number of colonies may or may not be significantly different when comparing plates from uninduced and induced bacteria (range between 1:1 – 1:100). In either case, you will still be able to find true recombinants with a high frequency. Analyze, say, 10-12 colonies by SpeI digestion of BAC miniprep DNA (see appendix C). Include a SpeI digest of the parent BAC as a control. Clones with a digestion pattern like the parent are likely to have undergone the desired mutation. Background clones (DOG resistant without the desired mutation) willl have obvious deletions, and should not be analyzed further. The clones with correct digestion pattern should be analyzed by PCR and sequencing of the mutated region. If the pattern is identical to the parental digestion pattern, it means that the bacteria most likely became DOG resistant due to the desired homologous recombination event. Alternatively, any deletion that contains the region with the galK cassette, but excludes the chloramphenicol region, will also be selected in the counterselection procedure. With a high frequency of homologous recombination as in he SW102 strain, the background is not likely to be a problem. In the unlikely event that too high a background is observed, try to increase the length of the homology arms, to increase the frequency of homologous recombination.

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