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Making LilrB2/PirB expression vectors

Start Date

Progress/condition/next steps

Completed? (Date)

Result

Location of part (if needed, I don't know)

Making pENTR from gBlocks:

We have:
Q1_LilrB2_Qx
Q1_PirB-N_Q2, Q2_PirB-C_Qx

We need:
pGGDONR : has Q1 and Qx sites, L1 and L2 sites

Golden-gating will result in:
pENTR_L1_LilrB2_L2 (order of L's correct, I checked!)
pENTR_L1_PirB2_L2

After that, Gibson (LR):
pENTR_L1_LilrB2_L2
pENTR_L4_Hef1a_R1 (not sure if this is the one we want)
pZDonor 1-GTW-2 (this is the DEST Shinjini used in her Geneious)

Same for PirB
pENTR_L1_PirB_L2
pENTR_L4_Hef1a_R1
pZDonor 1-GTW-2

We will get:

12_Hef1a_LilrB2
12_Hef1a_PirB

6/12/14

GoldenGate table:

Add	Conc	Volm(x)	LilrB2	PirB	Control	Order of adding
PirB-N	10ng/ul	5ul	0ul	5ul	0ul	2
PirB-C	10ng/ul	5ul	0ul	5ul	0ul	2
LilrB2	10ng/ul	5ul	5ul	0ul	0ul	2
GGDonr	44.8ng/ul	2.23ul	2.23ul	2.23ul	2.23ul	2
10x T4 Ligase Buffer		2ul	2ul	2ul	2ul	3
BSA		2ul	2ul	2ul	2ul	3
BsaI (enzyme)		1ul	1ul	1ul	1ul	4
T4 Ligase (enzyme)		1ul	1ul	1ul	1ul	3
H20			6.77ul	1.77ul	11.77ul	1

Total		20ul	20ul	20ul	20ul

1	LilrB2
2	PirB
3	Control

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