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From 2012.igem.org

I. Check Old Plates for Growth

• RESULTS: There was XL1-Blue colony growth on Kan50 plates from 15 Aug 2012 that was not present on controls for the 1:1 (~4 colonies) and 1:2 (~1 colony) vector-cassette ligations.

II. Colony PCR

• Mix Reagents:

• Mix according to the following table

	1 Reaction	Master Mix (x6)
Reagent	(uL)	(uL)
water	17.25	103.5
5x KAPA HiFi buffer	5	30
10 mM dNTP mix	0.75	4.5
10 uM primer (F)	0.75 (pBf)	4.5 (pBf)
10 uM primer (R)	0.75 (pBr)	4.5 (pBr)
Template	-	-
KAPA HiFi polymerase	0.5	3

• Aliquot 50 uL of mix to 6 PCR tubes

• Note: Last tube (control) had less than others

• For templates, use a pipette tip to touch a colony from one of the plates, dip it into a PCR tube, and then drop it into a culture tube as described in the next section (III). Do not use template for control.

• Run PCR

Step	Temp (oC)	Time
1	95	5 m
2	98	20 s
3	63	15 s
4	72	5 m
5	GOTO 2	34x
6	72	5 m
7	4	∞

III. Start Liquid Cultures for PCR Colonies

• Make LB-Kanamycin (50 ug/mL) media

• 10 mL of LB media
• 10 uL of Kanamycin stock (50 mg/mL)

• Aliquot ~2 mL of LB-Kanamycin media to 5 culture tubes

• Drop the pipette tip from each colony PCR into a tube labeled in the same way as the PCR tubes

• Incubate overnight with shaking at ~32 ^oC