User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/01/24 - Revision history

Behzad Damadzadeh: /* 01/24/2013 */

2013-02-20T19:13:46Z

01/24/2013

←Older revision		Revision as of 19:13, 20 February 2013
Line 35:		Line 35:
	Ligation of NLS-HIS-STOP and V0120 vector in DH5α-T and BL21		Ligation of NLS-HIS-STOP and V0120 vector in DH5α-T and BL21
	* V0120 cut with X/P and purified from the gel.		* V0120 cut with X/P and purified from the gel.
-	<div class="floatright">[[~~Image:~~Image:V0120(X-P).png ]]<br>V0120 Vector cut with Xba1 and Pst1	+	<div class="floatright">[[Image: V0120(X-P).png ]]<br>V0120 Vector cut with Xba1 and Pst1
	</div>		</div>
	* Ligations		* Ligations

Behzad Damadzadeh: /* 01/24/2013 */

2013-02-20T19:12:52Z

01/24/2013

←Older revision		Revision as of 19:12, 20 February 2013
Line 35:		Line 35:
	Ligation of NLS-HIS-STOP and V0120 vector in DH5α-T and BL21		Ligation of NLS-HIS-STOP and V0120 vector in DH5α-T and BL21
	* V0120 cut with X/P and purified from the gel.		* V0120 cut with X/P and purified from the gel.
		+	<div class="floatright">[[Image:Image:V0120(X-P).png ]]<br>V0120 Vector cut with Xba1 and Pst1
		+	</div>
	* Ligations		* Ligations
	{\| {{table}} cellspacing="3" <!-- Ligations table -->		{\| {{table}} cellspacing="3" <!-- Ligations table -->

Behzad Damadzadeh: /* 01/24/2013 */

2013-02-20T18:26:11Z

01/24/2013

←Older revision		Revision as of 18:26, 20 February 2013
Line 46:		Line 46:
	\| 3. E. coli BL-21 NLS-HIS-STOP/size, 25 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 2:1 No Colonies</font>		\| 3. E. coli BL-21 NLS-HIS-STOP/size, 25 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 2:1 No Colonies</font>
	\|-		\|-
-	\| 4. E. coli BL-21 NLS-HIS-STOP/size, 7.2 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 </font>	+	\| 4. E. coli BL-21 NLS-HIS-STOP/size, 7.2 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies </font>
	\|-		\|-
-	\| 5. E. coli BL-21 NLS-HIS-STOP/size, 7.2 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 </font>	+	\| 5. E. coli BL-21 NLS-HIS-STOP/size, 7.2 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font>
	\|-		\|-
-	\| 6. E. coli BL-21 NLS-HIS-STOP/size, 7.2 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 </font>	+	\| 6. E. coli BL-21 NLS-HIS-STOP/size, 7.2 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font>
	\|-		\|-
	\| 7. V0120 (X/P)/3200)/ 15 ng (Control Plate)\|\|		\| 7. V0120 (X/P)/3200)/ 15 ng (Control Plate)\|\|

Behzad Damadzadeh: /* 01/24/2013 */

2013-01-25T23:54:46Z

01/24/2013

←Older revision		Revision as of 23:54, 25 January 2013
Line 75:		Line 75:
	* The incubation time for the ligation process was 30min at room temperature.		* The incubation time for the ligation process was 30min at room temperature.
	* Reaction number 1, 30 μL DH5α-Turbo; ice 5 min.; plate on amp agar		* Reaction number 1, 30 μL DH5α-Turbo; ice 5 min.; plate on amp agar
-	* Reaction number 2, 3, 4, 30μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 45 sec.; add 800 μL SOC medium; shake @ 37°C 30 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar	+	* Reaction number 2, 3, 4, 5, 6, 7 30μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 45 sec.; add 800 μL SOC medium; shake @ 37°C 30 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar

Behzad Damadzadeh: /* 01/24/2013 */

2013-01-25T23:34:53Z

01/24/2013

Behzad Damadzadeh: /* 01/24/2013 */

2013-01-25T20:49:01Z

01/24/2013

←Older revision		Revision as of 20:49, 25 January 2013
Line 40:		Line 40:
	\| Ligation \|\| <font color="blue"><u></font>		\| Ligation \|\| <font color="blue"><u></font>
	\|-		\|-
-	\| 1. E. coli DH5α-T NLS-HIS-STOP/size, 20 ng +V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font>	+	\| 1. E. coli DH5α-T NLS-HIS-STOP/size, 25 ng +V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font>
	\|-		\|-
-	\| 2. E. coli BL-21 NLS-HIS-STOP/size, 16 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font>	+	\| 2. E. coli BL-21 NLS-HIS-STOP/size, 25 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font>
	\|-		\|-
-	\| 3. E. coli BL-21 NLS-HIS-STOP/size, ~~22.8~~ ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 2:1 No Colonies</font>	+	\| 3. E. coli BL-21 NLS-HIS-STOP/size, 25 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 2:1 No Colonies</font>
	\|-		\|-
-	\| 4. E. coli BL-21 NLS-HIS-STOP/size, 20 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 </font>	+	\| 4. E. coli BL-21 NLS-HIS-STOP/size, --- ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 </font>
	\|-		\|-
	\| 5. V0120 (X/P)/3200)/ 15 ng (Control Plate)\|\|		\| 5. V0120 (X/P)/3200)/ 15 ng (Control Plate)\|\|
Line 69:		Line 69:

	* The incubation time for the ligation process was 30min at room temperature.		* The incubation time for the ligation process was 30min at room temperature.
-	* ~~Fast transformation~~, 30 min on ice.	+	* Reaction number 1, 30 μL DH5α-Turbo; ice 5 min.; plate on amp agar
		+	* Reaction number 2, 3, 4, 30μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 90 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar

Behzad Damadzadeh: /* 01/24/2013 */

2013-01-25T20:43:36Z

01/24/2013

←Older revision		Revision as of 20:43, 25 January 2013
Line 9:		Line 9:
	=01/24/2013=		=01/24/2013=

-	'Making Standardized DNA Part (NLS-HIS-STOP)'	+	'''Making Standardized DNA Part (NLS-HIS-STOP)'''

	NLS-6His-STOP-F1 CTAGAcccaagaaaaagcgcaaggtacaccatcaccaccatcacgcgtaaagctgagACTAGTAGCGGCCGCTGCA 76		NLS-6His-STOP-F1 CTAGAcccaagaaaaagcgcaaggtacaccatcaccaccatcacgcgtaaagctgagACTAGTAGCGGCCGCTGCA 76
Line 40:		Line 40:
	\| Ligation \|\| <font color="blue"><u></font>		\| Ligation \|\| <font color="blue"><u></font>
	\|-		\|-
-	\| 1. E. coli DH5α-T NLS-HIS-STOP/size, 20 ng +V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">~~KNLS~~-HIS-STOP/C-His 3:1 No Colonies</font>	+	\| 1. E. coli DH5α-T NLS-HIS-STOP/size, 20 ng +V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font>
	\|-		\|-
	\| 2. E. coli BL-21 NLS-HIS-STOP/size, 16 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font>		\| 2. E. coli BL-21 NLS-HIS-STOP/size, 16 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font>
Line 55:		Line 55:
	\|   \|\| 1 \|\| 2 \|\| 3 \|\| 4 \|\| 5		\|   \|\| 1 \|\| 2 \|\| 3 \|\| 4 \|\| 5
	\|-		\|-
-	\| Insert DNA \|\| 2.34 \|\| 2.34 \|\| 2.05 \|\| ~~2.05~~ \|\| ---	+	\| Insert DNA \|\| 0.20 \|\| 0.20 \|\| 2.05 \|\| --- \|\| ---
	\|-		\|-
-	\| Vector DNA \|\| 1.92 \|\| 1.92 \|\| 1.92 \|\| ~~1.92~~ \|\| 1.92	+	\| Vector DNA \|\| 3.50 \|\| 3.50 \|\| 3.50 \|\| --- \|\| 3.50
	\|-		\|-
-	\| 2x lgn buf (Roche) \|\| 5.26 \|\| 5.26 \|\| 5.0 \|\| 5.0 \|\| 5.0	+	\| 2x lgn buf (Roche) \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0
	\|-		\|-
	\| T4 ligase (NEB) \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0		\| T4 ligase (NEB) \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0
	\|-		\|-
-	\| dH<sub>2</sub>O \|\| 0 \|\| 0 \|\| 0.03 \|\| 0.03 \|\| 2.08	+	\| dH<sub>2</sub>O \|\| 0 .30\|\| 0.30 \|\| 0.25 \|\| 0.03 \|\| 0.50
	\|-		\|-
-	\|   \|\| 10.52 μL \|\| 10.52 μL \|\| 10μl \|\| 10μL \|\| 10μL	+	\|   \|\| 10.00 μL \|\| 10.00 μL \|\| 10μl \|\| 10μL \|\| 10μL
	\|}		\|}

	* The incubation time for the ligation process was 30min at room temperature.		* The incubation time for the ligation process was 30min at room temperature.
	* Fast transformation, 30 min on ice.		* Fast transformation, 30 min on ice.

Behzad Damadzadeh: /* 01/24/2013 */

2013-01-25T20:39:16Z

01/24/2013

←Older revision		Revision as of 20:39, 25 January 2013
Line 12:		Line 12:

	NLS-6His-STOP-F1 CTAGAcccaagaaaaagcgcaaggtacaccatcaccaccatcacgcgtaaagctgagACTAGTAGCGGCCGCTGCA 76		NLS-6His-STOP-F1 CTAGAcccaagaaaaagcgcaaggtacaccatcaccaccatcacgcgtaaagctgagACTAGTAGCGGCCGCTGCA 76
		+
	NLS-6His-STOP-R1 GCGGCCGCTACTAGTctcagctttacgcgtgatggtggtgatggtgtaccttgcgctttttcttgggT 68		NLS-6His-STOP-R1 GCGGCCGCTACTAGTctcagctttacgcgtgatggtggtgatggtgtaccttgcgctttttcttgggT 68

Line 30:		Line 31:
	Heat at 100°C for 5 min., remove the entire heat block or water bath from the heat source, and allow to cool slowly to room temperature.<br>		Heat at 100°C for 5 min., remove the entire heat block or water bath from the heat source, and allow to cool slowly to room temperature.<br>
	--> *10x annealing buffer: 1 M NaCl; 100 mM Tris-HCl, pH 7.4		--> *10x annealing buffer: 1 M NaCl; 100 mM Tris-HCl, pH 7.4
		+
		+
		+	Ligation of NLS-HIS-STOP and V0120 vector in DH5α-T and BL21
		+	* V0120 cut with X/P and purified from the gel.
		+	* Ligations
		+	{\| {{table}} cellspacing="3" <!-- Ligations table -->
		+	\|- bgcolor=#cfcfcf
		+	\| Ligation \|\| <font color="blue"><u></font>
		+	\|-
		+	\| 1. E. coli DH5α-T NLS-HIS-STOP/size, 20 ng +V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">KNLS-HIS-STOP/C-His 3:1 No Colonies</font>
		+	\|-
		+	\| 2. E. coli BL-21 NLS-HIS-STOP/size, 16 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font>
		+	\|-
		+	\| 3. E. coli BL-21 NLS-HIS-STOP/size, 22.8 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 2:1 No Colonies</font>
		+	\|-
		+	\| 4. E. coli BL-21 NLS-HIS-STOP/size, 20 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 </font>
		+	\|-
		+	\| 5. V0120 (X/P)/3200)/ 15 ng (Control Plate)\|\|
		+	\|}
		+
		+	* Calculations are for the ng of insert we need to get a 3:1 ratio of insert molecules to 50 ng vector molecules
		+	{\| {{table}} cellspacing="3" <!-- Ligation rxn table -->
		+	\|   \|\| 1 \|\| 2 \|\| 3 \|\| 4 \|\| 5
		+	\|-
		+	\| Insert DNA \|\| 2.34 \|\| 2.34 \|\| 2.05 \|\| 2.05 \|\| ---
		+	\|-
		+	\| Vector DNA \|\| 1.92 \|\| 1.92 \|\| 1.92 \|\| 1.92 \|\| 1.92
		+	\|-
		+	\| 2x lgn buf (Roche) \|\| 5.26 \|\| 5.26 \|\| 5.0 \|\| 5.0 \|\| 5.0
		+	\|-
		+	\| T4 ligase (NEB) \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0
		+	\|-
		+	\| dH<sub>2</sub>O \|\| 0 \|\| 0 \|\| 0.03 \|\| 0.03 \|\| 2.08
		+	\|-
		+	\|   \|\| 10.52 μL \|\| 10.52 μL \|\| 10μl \|\| 10μL \|\| 10μL
		+	\|}
		+
		+	* The incubation time for the ligation process was 30min at room temperature.
		+	* Fast transformation, 30 min on ice.

Behzad Damadzadeh: /* Date */

2013-01-24T21:18:54Z

Date

←Older revision		Revision as of 21:18, 24 January 2013
Line 7:		Line 7:
	<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->

-	=~~Date~~=	+	=01/24/2013=

-	''~~'List title'''~~	+	'Making Standardized DNA Part (NLS-HIS-STOP)'
-	~~# List items~~	+
		+	NLS-6His-STOP-F1 CTAGAcccaagaaaaagcgcaaggtacaccatcaccaccatcacgcgtaaagctgagACTAGTAGCGGCCGCTGCA 76
		+	NLS-6His-STOP-R1 GCGGCCGCTACTAGTctcagctttacgcgtgatggtggtgatggtgtaccttgcgctttttcttgggT 68
		+
		+	Set up an annealing reaction as follows:
		+
		+	{\|
		+	\|-
		+	\| Sense oligo 1 (100 μM) \|\| 3.0 μl
		+	\|-
		+	\| Anti-sense oligo (100 μM) \|\| 3.0 μl
		+	\|-
		+	\| 10x annealing buffer* \|\| 2.0 μl
		+	\|-
		+	\| dH2O \|\| 12.0 μl
		+	\|-
		+	\|   \|\| 20 μl
		+	\|}
		+	Heat at 100°C for 5 min., remove the entire heat block or water bath from the heat source, and allow to cool slowly to room temperature.<br>
		+	--> *10x annealing buffer: 1 M NaCl; 100 mM Tris-HCl, pH 7.4

Behzad Damadzadeh: Autocreate 2013/01/24 Entry for User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli

2013-01-24T21:11:12Z

Autocreate 2013/01/24 Entry for User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli

New page

{|{{table}} width="800"
|-
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>      </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|-
| colspan="2"|


=Date=

'''List title'''
# List items

←Older revision		Revision as of 23:34, 25 January 2013
Line 46:		Line 46:
	\| 3. E. coli BL-21 NLS-HIS-STOP/size, 25 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 2:1 No Colonies</font>		\| 3. E. coli BL-21 NLS-HIS-STOP/size, 25 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 2:1 No Colonies</font>
	\|-		\|-
-	\| 4. E. coli BL-21 NLS-HIS-STOP/size, ~~---~~ ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 </font>	+	\| 4. E. coli BL-21 NLS-HIS-STOP/size, 7.2 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 </font>
	\|-		\|-
-	\| 5. V0120 (X/P)/3200)/ 15 ng (Control Plate)\|\|	+	\| 5. E. coli BL-21 NLS-HIS-STOP/size, 7.2 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 </font>
		+	\|-
		+	\| 6. E. coli BL-21 NLS-HIS-STOP/size, 7.2 ng + V0120 (X/P)/3200, 14.1ng \|\| <font color="blue">NLS-HIS-STOP/C-His 3:1 </font>
		+	\|-
		+	\| 7. V0120 (X/P)/3200)/ 15 ng (Control Plate)\|\|
	\|}		\|}

		+	* Reactions 4, 5, 6 the annealing process of the oligos were done in 94°C.
	* Calculations are for the ng of insert we need to get a 3:1 ratio of insert molecules to 50 ng vector molecules		* Calculations are for the ng of insert we need to get a 3:1 ratio of insert molecules to 50 ng vector molecules
	{\| {{table}} cellspacing="3" <!-- Ligation rxn table -->		{\| {{table}} cellspacing="3" <!-- Ligation rxn table -->
-	\|   \|\| 1 \|\| 2 \|\| 3 \|\| 4 \|\| 5	+	\|   \|\| 1 \|\| 2 \|\| 3 \|\| 4 \|\| 5 \|\| 6 \|\| 7
	\|-		\|-
-	\| Insert DNA \|\| 0.20 \|\| 0.20 \|\| 2.05 \|\| ~~---~~ \|\| ---	+	\| Insert DNA \|\| 0.20 \|\| 0.20 \|\| 2.05 \|\| 0.20 \|\| 0.5 \|\|0.70 \|\|---
	\|-		\|-
-	\| Vector DNA \|\| 3.50 \|\| 3.50 \|\| 3.50 \|\| ~~---~~ \|\| 3.50	+	\| Vector DNA \|\| 3.50 \|\| 3.50 \|\| 3.50 \|\| 3.50 \|\| 3.50\|\| 3.50 \|\| 3.50
	\|-		\|-
-	\| 2x lgn buf (Roche) \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0	+	\| 2x lgn buf (Roche) \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.2 \|\| 5.0
	\|-		\|-
-	\| T4 ligase (NEB) \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0	+	\| T4 ligase (NEB) \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0
	\|-		\|-
-	\| dH<sub>2</sub>O \|\| 0 .30\|\| 0.30 \|\| 0.25 \|\| 0.03 \|\| 0.50	+	\| dH<sub>2</sub>O \|\| 0 .30\|\| 0.30 \|\| 0.25 \|\| 0.30 \|\| 0.00 \|\| --- \|\| 0.50
	\|-		\|-
-	\|   \|\| 10.00 μL \|\| 10.00 μL \|\| 10μl \|\| 10μL \|\| 10μL	+	\|   \|\| 10.00 μL \|\| 10.00 μL \|\| 10μl \|\| 10μL \|\| 10μL \|\| 10μL \|\| 10μL
	\|}		\|}

	* The incubation time for the ligation process was 30min at room temperature.		* The incubation time for the ligation process was 30min at room temperature.
	* Reaction number 1, 30 μL DH5α-Turbo; ice 5 min.; plate on amp agar		* Reaction number 1, 30 μL DH5α-Turbo; ice 5 min.; plate on amp agar
-	* Reaction number 2, 3, 4, 30μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 90 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar	+	* Reaction number 2, 3, 4, 30μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 45 sec.; add 800 μL SOC medium; shake @ 37°C 30 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar