Bac vector library - Revision history

imported>Ctelmer: /* BAC Recombineering */

2020-05-21T01:38:23Z

BAC Recombineering

← Older revision		Revision as of 01:38, 21 May 2020
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	== BAC Recombineering ==		== BAC Recombineering ==

	<p>More information is available here: [https://pubmed.ncbi.nlm.nih.gov/30948008/?from_term=buckley+k&from_page=2&from_pos=3 ~~Techniques for analyzing gene expression using BAC-based reporter constructs.~~ Buckley KM, Ettensohn CA~~. Methods Cell Biol. 2019;151:197-218. doi: 10.1016/bs.mcb.2019.01.004. Epub 2019 Feb 23. PMID: 30948008 Review~~.]</p>		<p>More information is available here: [https://pubmed.ncbi.nlm.nih.gov/30948008/?from_term=buckley+k&from_page=2&from_pos=3 Buckley KM, Ettensohn CA.]</p>
	<br>		<br>
	<p><u>Materials</u>:</p>		<p><u>Materials</u>:</p>

imported>Ctelmer: /* BAC Recombineering */

2020-05-21T01:36:16Z

BAC Recombineering

← Older revision		Revision as of 01:36, 21 May 2020
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	<br>		<br>
	<p><u>Procedure:</u></p>		<p><u>Procedure:</u></p>
			<br>
	<p>''Prepare the recombination cassette''</p>		<p>''Prepare the recombination cassette''</p>
	<p>Recommendations for designing the “recombination arms” are available in [https://pubmed.ncbi.nlm.nih.gov/30948008/?from_term=buckley+k&from_page=2&from_pos=3 Buckley KM, Ettensohn, CA]</p>		<p>Recommendations for designing the “recombination arms” are available in [https://pubmed.ncbi.nlm.nih.gov/30948008/?from_term=buckley+k&from_page=2&from_pos=3 Buckley KM, Ettensohn, CA]</p>

imported>Ctelmer: /* BAC Recombineering */

2020-05-21T01:35:38Z

BAC Recombineering

← Older revision		Revision as of 01:35, 21 May 2020
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	<br>		<br>
	<p><u>Procedure:</u></p>		<p><u>Procedure:</u></p>
	~~<br>~~
	<p>''Prepare the recombination cassette''</p>		<p>''Prepare the recombination cassette''</p>

	<p>Recommendations for designing the “recombination arms” are available in [https://pubmed.ncbi.nlm.nih.gov/30948008/?from_term=buckley+k&from_page=2&from_pos=3 Buckley KM, Ettensohn, CA]</p>		<p>Recommendations for designing the “recombination arms” are available in [https://pubmed.ncbi.nlm.nih.gov/30948008/?from_term=buckley+k&from_page=2&from_pos=3 Buckley KM, Ettensohn, CA]</p>
	<br>		<br>

imported>Ctelmer: /* BAC Vector for Macro-Array Genomic Libraries */

2020-05-21T01:21:40Z

BAC Vector for Macro-Array Genomic Libraries

← Older revision		Revision as of 01:21, 21 May 2020
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	== BAC Vector for Macro-Array Genomic Libraries ==		== BAC Vector for Macro-Array Genomic Libraries ==

	Frengen E., Weichenhan D., Zhao B., Osoegawa K., van Geel M., de Jong P. J. 1999. A modular, positive selection bacterial artificial chromosome vector with multiple cloning sites. Genomics. 58(3):250-3. ~~[https://www.ncbi.nlm.nih.gov/pubmed/?term=10373322%5BUID%5D&utm_source=gquery&utm_medium=search PUBMED 10373322~~]		[https://www.ncbi.nlm.nih.gov/pubmed/?term=10373322%5BUID%5D&utm_source=gquery&utm_medium=search Frengen E., Weichenhan D., Zhao B., Osoegawa K., van Geel M., de Jong P. J. 1999. A modular, positive selection bacterial artificial chromosome vector with multiple cloning sites. Genomics. 58(3):250-3.]
	<br>		<br>
	<p><u>Abstract</u></p>		<p><u>Abstract</u></p>

imported>Ctelmer: /* BAC Library Screening */

2020-05-21T01:17:41Z

BAC Library Screening

← Older revision		Revision as of 01:17, 21 May 2020
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	<p>'''EXAMPLE:''' On the accompanying figure there is a positive spot pair circled in white. It lies in the left-middle sub-field at X-Y position I-10. That is I blocks (9) up from the bottom of the sub-field and 10 blocks over from the centerline. Thus the well position with in the plate is I-10. For this sub-field, the angle of the spot pair within the block indicates #31. There fore, the clone is located on plate #31 in well I-10.</p>		<p>'''EXAMPLE:''' On the accompanying figure there is a positive spot pair circled in white. It lies in the left-middle sub-field at X-Y position I-10. That is I blocks (9) up from the bottom of the sub-field and 10 blocks over from the centerline. Thus the well position with in the plate is I-10. For this sub-field, the angle of the spot pair within the block indicates #31. There fore, the clone is located on plate #31 in well I-10.</p>
	<p>'''NOTES:''' Identification of spot coordinates in the case where the background on the filter is very low is aided by pre-marking the filters when they are dry and the colony residue is visible. Dry filters are marked by indentations from a ball point pen. The pen tip is pressed into a filter that is placed on a piece of 3MM paper on a hard surface while observing the operation in oblique lighting. Dots can thus be made at the boundaries of the 6 sub-fields and at the extreme corners of the array.</p>		<p>'''NOTES:''' Identification of spot coordinates in the case where the background on the filter is very low is aided by pre-marking the filters when they are dry and the colony residue is visible. Dry filters are marked by indentations from a ball point pen. The pen tip is pressed into a filter that is placed on a piece of 3MM paper on a hard surface while observing the operation in oblique lighting. Dots can thus be made at the boundaries of the 6 sub-fields and at the extreme corners of the array.</p>
	To aid orientation in the newer filter sets, the A1 well for each plate has been left empty. Thus there are six empty squares that have no bacteria on the filter. After hybridization these squares have lower background and will orient the filter. The squares are 3 sets of 2 across when the label is on the upper right hand edge.</p>		<p>To aid orientation in the newer filter sets, the A1 well for each plate has been left empty. Thus there are six empty squares that have no bacteria on the filter. After hybridization these squares have lower background and will orient the filter. The squares are 3 sets of 2 across when the label is on the upper right hand edge.</p>

	== BAC Miniprep Protocol ==		== BAC Miniprep Protocol ==

imported>Ctelmer: /* BAC Recombineering */

2020-04-22T21:45:56Z

BAC Recombineering

imported>Ctelmer: /* BAC Recombineering */

2020-04-22T21:43:33Z

BAC Recombineering

← Older revision		Revision as of 21:43, 22 April 2020
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	<p>15. To verify that the transformation worked correctly, mini-prep the BACs, digest with NotI and analyze on a PFGE.</p>		<p>15. To verify that the transformation worked correctly, mini-prep the BACs, digest with NotI and analyze on a PFGE.</p>
	<p>16. Make glycerol stocks of positive EL250-BAC clones.</p>		<p>16. Make glycerol stocks of positive EL250-BAC clones.</p>
	Recombineer the fluorescent protein into the BAC DNA		<br>
			<p>''Recombineer the fluorescent protein into the BAC DNA''</p>
			<p>1. Pick a starter culture of EL250-BAC cells from frozen stocks or fresh streak in 3 mL LB/chl and incubate overnight at 30°C.</p>
			<p>2. Dilute the culture 1 mL in 50 mL fresh LB/chl and incubate for ~6 hrs at 30°C, until O.D.600 is 0.8-1.0.</p>
			<p>3. Place in pre-warmed 42°C shaking water bath for 15 min to activate the recombinase genes.</p>
			<p>If a shaking water bath is not available, gently swirl the flasks by hand for the entire 15 min, taking care to keep the bottom fully submerged in the bath. Due to this step (and the washing steps thereafter), it is difficult to prepare more than two BACs simultaneously.</p>
			<p>4. Immediately chill by swirling in ice-water slurry for ~10 min.</p>
			<p>5. Transfer to a pre-chilled 50 mL conical tube, pellet and wash as described above (on ice).</p>
			<p>6. Add 200 ng of FP cassette DNA to 10 μl electrocompetent EL250-BAC cells + 10 μl dH2O on ice.</p>
			<p>7. Transfer to ice-cold 0.1 mm electroporation cuvette. Electroporate at 1.8 kV/cm and immediately transfer to 1 mL S.O.C or LB. Incubate at 30°C for 1 hr. Plate 50 μl and 500 μl on LB chl/kan plates and incubate 24 hrs at 30°C.</p>
			<p>8. Colonies picked right after overnight incubation @ 30°C have free FP cassette in them, whereas those picked after a full 24 hr of incubation have recombined FP into the BAC. An EL250 cell with recombined BAC may grow slower due to low copy number of the BAC vector.</p>
			<p>9. Check the recombination by restriction digest or PCR.</p>
			<p>10. Make glycerol stocks (15%) of EL250-BAC/FP clones.</p>
			<p>''Remove the kanamycin cassette''</p>

	~~1. Pick a starter culture of EL250-BAC cells from frozen stocks or fresh streak in 3 mL LB/chl and incubate overnight at 30°C.~~		<p>1. Start an overnight culture of EL250-BAC/FP cells in 3 mL LB chl/kan. Dilute culture 1:50 in 25 mL of LB/chl. Grow at 30°C for ~5 hrs until O.D.600 is ~0.5.</p>
	~~2. Dilute the culture 1 mL in 50 mL fresh LB/chl and incubate for ~6 hrs at 30°C, until O.D.600 is 0.8-1.0.~~		<p>2. Add 0.25 mL 10% L-(+)-arabinose and grow for 1 hr at 30°C. **'''Note''': D-(-)- arabinose will not work. It has been tried.</p>
	~~3. Place in pre-warmed 42°C shaking water bath for 15 min to activate the recombinase genes.~~		<p>3. Dilute culture 1:10 in 10 mL of LB/chl and grow for 1 hr at at 30°C.</p>
	If a shaking water bath is not available, gently swirl the flasks by hand for the entire 15 min, taking care to keep the bottom fully submerged in the bath. Due to this step (and the washing steps thereafter), it is difficult to prepare more than two BACs simultaneously.		<p>4. Streak 2 μl of this culture on an LB/chl plate. Grow overnight at 30°C.</p>
	~~4. Immediately chill by swirling in ice-water slurry for ~10 min.~~		<p>5. Select colonies that have lost the kanamycin resistance cassette by streaking clones (~4) onto both LB/chl and LB/chl/kan plates and grow at 30°C overnight. The colonies that do not grow on the LB/chl/kan plates have successfully eliminated (flipped) the kanamycin resistance cassette. This step is very efficient and gives nearly 100% flipped clones.</p>
	~~5. Transfer to a pre-chilled 50 mL conical tube, pellet and wash as described above (on ice).~~		<p>6. PCR diagnostic and sequencing can be used to confirm removal of kan cassette.</p>
	~~6. Add 200 ng of FP cassette DNA to 10 μl electrocompetent EL250-BAC cells + 10 μl dH2O on ice.~~		<p>7. Make glycerol stocks (15% glycerol) of EL250-BAC/FP/kan-flipped clones.</p>
	7. Transfer to ice-cold 0.1 mm electroporation cuvette. Electroporate at 1.8 kV/cm and immediately transfer to 1 mL S.O.C or LB. Incubate at 30°C for 1 hr. Plate 50 μl and 500 μl on LB chl/kan plates and incubate 24 hrs at 30°C.		<p>''Transform the recombinant BAC into DH10B cells''</p>
	8. Colonies picked right after overnight incubation @ 30°C have free FP cassette in them, whereas those picked after a full 24 hr of incubation have recombined FP into the BAC. An EL250 cell with recombined BAC may grow slower due to low copy number of the BAC vector.		<p>1. Miniprep an overnight culture of EL250-BAC/FP/kan-flipped cells.</p>
	~~9. Check the recombination by restriction digest or PCR.~~		<p>2. On ice, combine 1 µl of the BAC DNA (miniprep) with 10 µl commercial DH10B electrocompetent cells + 10 µl dH2O.</p>
	~~10. Make glycerol stocks (15%) of EL250-BAC/FP clones.~~		<p>3. Transfer to ice-cold 0.1mm electroporation cuvette. Electroporate at 1.4 kV/cm and immediately transfer to 1 mL S.O.C or LB.</p>
	~~Remove the kanamycin cassette~~		<p>4.Incubate at 37°C for 1 hr. Plate 50 µl and 200 µl on each of two LB/chl plates and incubate overnight at 37°C.</p>
			<p>5. Pick a few colonies in 3 mL LB/chl and grow overnight at 37°C.</p>
	1. Start an overnight culture of EL250-BAC/FP cells in 3 mL LB chl/kan. Dilute culture 1:50 in 25 mL of LB/chl. Grow at 30°C for ~5 hrs until O.D.600 is ~0.5.		<p>6. Miniprep these clones to confirm the size of the BAC insert and the FP recombination. Compare with the wildtype and non-flipped FP BACs as control. Analyze by PFGE.</p>
	2. Add 0.25 mL 10% L-(+)-arabinose and grow for 1 hr at 30°C. **Note: D-(-)- arabinose will not work. It has been tried.		<p>7. Make glycerol stocks (15% glycerol) of this construct.</p>
	3. Dilute culture 1:10 in 10 mL of LB/chl and grow for 1 hr at at 30°C.
	4. Streak 2 μl of this culture on an LB/chl plate. Grow overnight at 30°C.
	5. Select colonies that have lost the kanamycin resistance cassette by streaking clones (~4) onto both LB/chl and LB/chl/kan plates and grow at 30°C overnight. The colonies that do not grow on the LB/chl/kan plates have successfully eliminated (flipped) the kanamycin resistance cassette. This step is very efficient and gives nearly 100% flipped clones.
	6. PCR diagnostic and sequencing can be used to confirm removal of kan cassette.
	7. Make glycerol stocks (15% glycerol) of EL250-BAC/FP/kan-flipped clones.
	Transform the recombinant BAC into DH10B cells

	1. Miniprep an overnight culture of EL250-BAC/FP/kan-flipped cells.
	2. On ice, combine 1 µl of the BAC DNA (miniprep) with 10 µl commercial DH10B electrocompetent cells + 10 µl dH2O.
	3. Transfer to ice-cold 0.1mm electroporation cuvette. Electroporate at 1.4 kV/cm and immediately transfer to 1 mL S.O.C or LB.
	4.Incubate at 37°C for 1 hr. Plate 50 µl and 200 µl on each of two LB/chl plates and incubate overnight at 37°C.
	5. Pick a few colonies in 3 mL LB/chl and grow overnight at 37°C.
	6. Miniprep these clones to confirm the size of the BAC insert and the FP recombination. Compare with the wildtype and non-flipped FP BACs as control. Analyze by PFGE.
	7. Make glycerol stocks (15% glycerol) of this construct.

imported>Ctelmer: /* BAC Recombineering */

2020-04-22T21:38:13Z

BAC Recombineering

← Older revision		Revision as of 21:38, 22 April 2020
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	<p>1. Electrocompetent DH10B</p>		<p>1. Electrocompetent DH10B</p>
	<p>2. EL250. A DH10B-derived strain that contains a λ prophage with the recombination genes ''exo'', ''bet'', and ''gam''. These genes are repressed by the temperature-sensitive repressor cI857.</p>		<p>2. EL250. A DH10B-derived strain that contains a λ prophage with the recombination genes ''exo'', ''bet'', and ''gam''. These genes are repressed by the temperature-sensitive repressor cI857.</p>
			<br>
			<p><u>Procedure:</u></p>
			<br>
			<p>''Prepare the recombination cassette''</p>

			<p>Recommendations for designing the “recombination arms” are available in [https://pubmed.ncbi.nlm.nih.gov/30948008/?from_term=buckley+k&from_page=2&from_pos=3 Buckley KM, Ettensohn, CA]</p>
			<br>
			<p>''Amplify the recombination cassette (RC)''</p>

			<p>1. Use high fidelity DNA polymerase to amplify the RC with both arms (amplify from the 5′ end of the 5′ arm to the 3′ end of the 3′ arm) from plasmid DNA. Minimize the amount of plasmid DNA used in PCR.</p>
			<p>2. Run the product on an 0.8% agarose gel and purify the fragment using a commercial gel extraction kit.</p>
			<p>3. Treat the amplified RC with 5 U DpnI in the appropriate buffer.</p>
			<p>4. Incubate the reaction at 37<sup>o</sup>C for 1 hour.</p>
			<p>5. Heat the reaction at 65<sup>o</sup>C for 15 minutes.</p>
			<p>6. Precipitate the recombination cassette by adding 0.1 volume sodium acetate (3 M, pH 5.2) and 2 volumes cold ethanol (100%).</p>
			<p>7. Incubate at -20<sup>o</sup>C at least two hours to overnight.</p>
			<p>8. Pellet the DNA by centrifugation (maximum speed, 4<sup>o</sup>C, 30 minutes).</p>
			<p>9. Wash the pellet with 70% ethanol, dry briefly and resuspend in 20 μl H 2 O.</p>
			<br>
			<p>''Transform the BAC into EL250 cells''</p>
			<p>1. Pick a starter culture of EL250 cells from frozen stocks or fresh streak in 3mL LB (no antibiotic). Incubate shaking overnight at 30-32°C.</p>
			<p>2. Dilute the culture 0.7 mL in 50 mL fresh LB. Incubate for ~5 hrs at 30°C, until O.D.600 is 0.8-1.0. From this point on, KEEP ON ICE AT ALL TIMES.</p>
			<p>3. Centrifuge bacteria for 10 min, 4°C, 3000 rpm using a pre-chilled 50 mL conical.</p>
			<p>4. Resuspend in 50 mL ice-cold dH2O and spin down as above.</p>
			<p>Tip: resuspend the pellet first in 1 mL, but avoid pipetting. Instead rock/shake the tube briskly against and within the ice bucket. This takes time, but is necessary to better preserve the cells. Once the pellet is resuspended in a small volume, top off to 50 mL and mix gently by inverting a few times.</p>
			<p>5. Repeat steps 3 – 4.</p>
			<p>6. Centrifuge bacteria for 10 min, 4°C, 3000 rpm. Resuspend in 1 mL ice-cold dH2O as above. Transfer to chilled 1.5 mL tube.</p>
			<p>7. Centrifuge for 2 min, 4°C, maximum speed.</p>
			<p>8. Wash 3 times with ice-cold dH2O.</p>
			<p>9. Use the cells immediately for electroporation. Washed, electrocompetent cells can be frozen @ -80°C with 10% f.c. glycerin, but transforming efficiency will decrease. It is best to use fresh cells every time.</p>
			<p>10. Add 200 ng of BAC DNA (~1/2-1/3 of a mini-prep) to 10 μl electrocompetent EL250 cells + 10 μl dH2O on ice. Transfer to ice-cold 0.1 mm electroporation cuvette.</p>
			<p>11. Electroporate at 1.4-1.7 kV/cm, immediately add 1 mL S.O.C. or LB to the cuvette, and transfer to a 15 mL culture tube. BAC transformation work better at slightly lower voltages (1.4-1.7 kV/cm). The bigger the BAC, the lower the kV/cm.</p>
			<p>12. Incubate shaking at 30°C for 90 min.</p>
			<p>13. Plate everything on LB/chl plates and incubate up to 24 hrs at 30°C. Using the large plates increases quality of colonies and overall efficiency.</p>
			<p>14, Pick a few colonies and grow at 30°C overnight to 24 hr in LB/chl.</p>
			<p>15. To verify that the transformation worked correctly, mini-prep the BACs, digest with NotI and analyze on a PFGE.</p>
			<p>16. Make glycerol stocks of positive EL250-BAC clones.</p>
			Recombineer the fluorescent protein into the BAC DNA

			1. Pick a starter culture of EL250-BAC cells from frozen stocks or fresh streak in 3 mL LB/chl and incubate overnight at 30°C.
			2. Dilute the culture 1 mL in 50 mL fresh LB/chl and incubate for ~6 hrs at 30°C, until O.D.600 is 0.8-1.0.
			3. Place in pre-warmed 42°C shaking water bath for 15 min to activate the recombinase genes.
			If a shaking water bath is not available, gently swirl the flasks by hand for the entire 15 min, taking care to keep the bottom fully submerged in the bath. Due to this step (and the washing steps thereafter), it is difficult to prepare more than two BACs simultaneously.
			4. Immediately chill by swirling in ice-water slurry for ~10 min.
			5. Transfer to a pre-chilled 50 mL conical tube, pellet and wash as described above (on ice).
			6. Add 200 ng of FP cassette DNA to 10 μl electrocompetent EL250-BAC cells + 10 μl dH2O on ice.
			7. Transfer to ice-cold 0.1 mm electroporation cuvette. Electroporate at 1.8 kV/cm and immediately transfer to 1 mL S.O.C or LB. Incubate at 30°C for 1 hr. Plate 50 μl and 500 μl on LB chl/kan plates and incubate 24 hrs at 30°C.
			8. Colonies picked right after overnight incubation @ 30°C have free FP cassette in them, whereas those picked after a full 24 hr of incubation have recombined FP into the BAC. An EL250 cell with recombined BAC may grow slower due to low copy number of the BAC vector.
			9. Check the recombination by restriction digest or PCR.
			10. Make glycerol stocks (15%) of EL250-BAC/FP clones.
			Remove the kanamycin cassette

			1. Start an overnight culture of EL250-BAC/FP cells in 3 mL LB chl/kan. Dilute culture 1:50 in 25 mL of LB/chl. Grow at 30°C for ~5 hrs until O.D.600 is ~0.5.
			2. Add 0.25 mL 10% L-(+)-arabinose and grow for 1 hr at 30°C. **Note: D-(-)- arabinose will not work. It has been tried.
			3. Dilute culture 1:10 in 10 mL of LB/chl and grow for 1 hr at at 30°C.
			4. Streak 2 μl of this culture on an LB/chl plate. Grow overnight at 30°C.
			5. Select colonies that have lost the kanamycin resistance cassette by streaking clones (~4) onto both LB/chl and LB/chl/kan plates and grow at 30°C overnight. The colonies that do not grow on the LB/chl/kan plates have successfully eliminated (flipped) the kanamycin resistance cassette. This step is very efficient and gives nearly 100% flipped clones.
			6. PCR diagnostic and sequencing can be used to confirm removal of kan cassette.
			7. Make glycerol stocks (15% glycerol) of EL250-BAC/FP/kan-flipped clones.
			Transform the recombinant BAC into DH10B cells

			1. Miniprep an overnight culture of EL250-BAC/FP/kan-flipped cells.
			2. On ice, combine 1 µl of the BAC DNA (miniprep) with 10 µl commercial DH10B electrocompetent cells + 10 µl dH2O.
			3. Transfer to ice-cold 0.1mm electroporation cuvette. Electroporate at 1.4 kV/cm and immediately transfer to 1 mL S.O.C or LB.
			4.Incubate at 37°C for 1 hr. Plate 50 µl and 200 µl on each of two LB/chl plates and incubate overnight at 37°C.
			5. Pick a few colonies in 3 mL LB/chl and grow overnight at 37°C.
			6. Miniprep these clones to confirm the size of the BAC insert and the FP recombination. Compare with the wildtype and non-flipped FP BACs as control. Analyze by PFGE.
			7. Make glycerol stocks (15% glycerol) of this construct.

imported>Ctelmer at 21:24, 22 April 2020

2020-04-22T21:24:00Z

← Older revision		Revision as of 21:24, 22 April 2020
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	<p><u>Materials</u>:</p>		<p><u>Materials</u>:</p>
	<p>''Reagents'':</p>		<p>''Reagents'':</p>
	<p>LB with kanamycin (25 μg/mL)</p>		<p style="margin-left:10%; margin-right:10%;">LB with kanamycin (25 μg/mL)</p>
	<p>LB with chloramphenicol (12.5 μg/mL)</p>		<p style="margin-left:10%; margin-right:10%;">LB with chloramphenicol (12.5 μg/mL)</p>
	<p>SOC media</p>		<p style="margin-left:10%; margin-right:10%;">SOC media</p>
	<p>10% L-(+)-arabinose</p>		<p style="margin-left:10%; margin-right:10%;">10% L-(+)-arabinose</p>
	<p>Gel extraction kit</p>		<p style="margin-left:10%; margin-right:10%;">Gel extraction kit</p>
	<p>High fidelity DNA polymerase</p>		<p style="margin-left:10%; margin-right:10%;">High fidelity DNA polymerase</p>
	<p>DpnI</p>		<p style="margin-left:10%; margin-right:10%;">DpnI</p>
	<p>3 M NaOAc (pH 5.2)</p>		<p style="margin-left:10%; margin-right:10%;">3 M NaOAc (pH 5.2)</p>
	<p>''Cell lines'':</p>		<p>''Cell lines'':</p>
	<p>1. Electrocompetent DH10B</p>		<p>1. Electrocompetent DH10B</p>
	<p>2. EL250. A DH10B-derived strain that contains a λ prophage with the recombination genes ''exo'', ''bet'', and ''gam''. These genes are repressed by the temperature-sensitive repressor cI857.</p>		<p>2. EL250. A DH10B-derived strain that contains a λ prophage with the recombination genes ''exo'', ''bet'', and ''gam''. These genes are repressed by the temperature-sensitive repressor cI857.</p>

imported>Ctelmer: /* BAC Recombineering */

2020-04-22T21:22:11Z

BAC Recombineering

← Older revision		Revision as of 21:22, 22 April 2020
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	<p>More information is available here: [https://pubmed.ncbi.nlm.nih.gov/30948008/?from_term=buckley+k&from_page=2&from_pos=3 Techniques for analyzing gene expression using BAC-based reporter constructs. Buckley KM, Ettensohn CA. Methods Cell Biol. 2019;151:197-218. doi: 10.1016/bs.mcb.2019.01.004. Epub 2019 Feb 23. PMID: 30948008 Review.]</p>		<p>More information is available here: [https://pubmed.ncbi.nlm.nih.gov/30948008/?from_term=buckley+k&from_page=2&from_pos=3 Techniques for analyzing gene expression using BAC-based reporter constructs. Buckley KM, Ettensohn CA. Methods Cell Biol. 2019;151:197-218. doi: 10.1016/bs.mcb.2019.01.004. Epub 2019 Feb 23. PMID: 30948008 Review.]</p>
			<br>
			<p><u>Materials</u>:</p>
			<p>''Reagents'':</p>
			<p>LB with kanamycin (25 μg/mL)</p>
			<p>LB with chloramphenicol (12.5 μg/mL)</p>
			<p>SOC media</p>
			<p>10% L-(+)-arabinose</p>
			<p>Gel extraction kit</p>
			<p>High fidelity DNA polymerase</p>
			<p>DpnI</p>
			<p>3 M NaOAc (pH 5.2)</p>
			<p>''Cell lines'':</p>
			<p>1. Electrocompetent DH10B</p>
			<p>2. EL250. A DH10B-derived strain that contains a λ prophage with the recombination genes ''exo'', ''bet'', and ''gam''. These genes are repressed by the temperature-sensitive repressor cI857.</p>

← Older revision		Revision as of 21:45, 22 April 2020
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	<p>''Amplify the recombination cassette (RC)''</p>		<p>''Amplify the recombination cassette (RC)''</p>

	<p>1. Use high fidelity DNA polymerase to amplify the RC with both arms (amplify from the 5′ end of the 5′ arm to the 3′ end of the 3′ arm) from plasmid DNA. Minimize the amount of plasmid DNA used in PCR.</p>		<p>1. Use high fidelity DNA polymerase to amplify the RC with both arms (amplify from the 5′ end of the 5′ arm to the 3′ end of the 3′ arm) from plasmid DNA. Minimize the amount of plasmid DNA used in PCR.</p>
	<p>2. Run the product on an 0.8% agarose gel and purify the fragment using a commercial gel extraction kit.</p>		<p>2. Run the product on an 0.8% agarose gel and purify the fragment using a commercial gel extraction kit.</p>
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	<p>''Transform the BAC into EL250 cells''</p>		<p>''Transform the BAC into EL250 cells''</p>
	<p>1. Pick a starter culture of EL250 cells from frozen stocks or fresh streak in 3mL LB (no antibiotic). Incubate shaking overnight at 30-32°C.</p>		<p>1. Pick a starter culture of EL250 cells from frozen stocks or fresh streak in 3mL LB (no antibiotic). Incubate shaking overnight at 30-32°C.</p>
	<p>2. Dilute the culture 0.7 mL in 50 mL fresh LB. Incubate for ~5 hrs at 30°C, until O.D.600 is 0.8-1.0. From this point on, KEEP ON ICE AT ALL TIMES.</p>		<p>2. Dilute the culture 0.7 mL in 50 mL fresh LB. Incubate for ~5 hrs at 30°C, until O.D.600 is 0.8-1.0. From this point on, '''KEEP ON ICE AT ALL TIMES.'''</p>
	<p>3. Centrifuge bacteria for 10 min, 4°C, 3000 rpm using a pre-chilled 50 mL conical.</p>		<p>3. Centrifuge bacteria for 10 min, 4°C, 3000 rpm using a pre-chilled 50 mL conical.</p>
	<p>4. Resuspend in 50 mL ice-cold dH2O and spin down as above.</p>		<p>4. Resuspend in 50 mL ice-cold dH2O and spin down as above.</p>
	<p>Tip: resuspend the pellet first in 1 mL, but avoid pipetting. Instead rock/shake the tube briskly against and within the ice bucket. This takes time, but is necessary to better preserve the cells. Once the pellet is resuspended in a small volume, top off to 50 mL and mix gently by inverting a few times.</p>		<p>'''Tip''': resuspend the pellet first in 1 mL, but avoid pipetting. Instead rock/shake the tube briskly against and within the ice bucket. This takes time, but is necessary to better preserve the cells. Once the pellet is resuspended in a small volume, top off to 50 mL and mix gently by inverting a few times.</p>
	<p>5. Repeat steps 3 – 4.</p>		<p>5. Repeat steps 3 – 4.</p>
	<p>6. Centrifuge bacteria for 10 min, 4°C, 3000 rpm. Resuspend in 1 mL ice-cold dH2O as above. Transfer to chilled 1.5 mL tube.</p>		<p>6. Centrifuge bacteria for 10 min, 4°C, 3000 rpm. Resuspend in 1 mL ice-cold dH2O as above. Transfer to chilled 1.5 mL tube.</p>
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	<p>9. Check the recombination by restriction digest or PCR.</p>		<p>9. Check the recombination by restriction digest or PCR.</p>
	<p>10. Make glycerol stocks (15%) of EL250-BAC/FP clones.</p>		<p>10. Make glycerol stocks (15%) of EL250-BAC/FP clones.</p>
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	<p>''Remove the kanamycin cassette''</p>		<p>''Remove the kanamycin cassette''</p>

	<p>1. Start an overnight culture of EL250-BAC/FP cells in 3 mL LB chl/kan. Dilute culture 1:50 in 25 mL of LB/chl. Grow at 30°C for ~5 hrs until O.D.600 is ~0.5.</p>		<p>1. Start an overnight culture of EL250-BAC/FP cells in 3 mL LB chl/kan. Dilute culture 1:50 in 25 mL of LB/chl. Grow at 30°C for ~5 hrs until O.D.600 is ~0.5.</p>
	<p>2. Add 0.25 mL 10% L-(+)-arabinose and grow for 1 hr at 30°C. **'''Note''': D-(-)- arabinose will not work. It has been tried.</p>		<p>2. Add 0.25 mL 10% L-(+)-arabinose and grow for 1 hr at 30°C. **'''Note''': D-(-)- arabinose will not work. It has been tried.</p>
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	<p>6. PCR diagnostic and sequencing can be used to confirm removal of kan cassette.</p>		<p>6. PCR diagnostic and sequencing can be used to confirm removal of kan cassette.</p>
	<p>7. Make glycerol stocks (15% glycerol) of EL250-BAC/FP/kan-flipped clones.</p>		<p>7. Make glycerol stocks (15% glycerol) of EL250-BAC/FP/kan-flipped clones.</p>
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	<p>''Transform the recombinant BAC into DH10B cells''</p>		<p>''Transform the recombinant BAC into DH10B cells''</p>
	<p>1. Miniprep an overnight culture of EL250-BAC/FP/kan-flipped cells.</p>		<p>1. Miniprep an overnight culture of EL250-BAC/FP/kan-flipped cells.</p>