Tasks for This Week

Protein Expression Screening

#	Clone ID	Name	Induction	His Tag	pI MW	Expression
1	2596	pT7A30-D7324-6H-CCMV			9.89 / 23.4
2	2586	pET28a-6H-D7324-AP205			9.88 / 17.6
3	2595	pDB.His.GST-D7324			6.90 / 31
4	2594	pDB.His.GST-SpyCatcher			5.75 / 39.3
5	2593	pDB.His.GST-SpyCatcher-AP205			5.99 / 53.6
6	2585	pT7A3-TRX-multi-Tags			4.67 / 49.7
7	2587	pT7A3-GST-multi-Tags			4.97 / 63.9
8	2604	pCold-MBP-multitag			4.88 / 80.6
9	2580	pET28a-Qbeta-Tat-6His			10.35 / 18.7
10	2579	pET28a-6H-Tat-AP205			10.54 / 18.3
11	129	pT7A30-TthMuts			5.87 / 91.8
12	136	pL6H-Tat-Cre			10.30 / 42.7
13	135	pQE-80L-HTNCre			10.30 / 42.8
14	170	pT7-MBP-(tev)-GST-(3c)-Tat-NLS-iFLP-6H			8.64 / 123.3
15	169	pBad-MBP-(Tev)-GST-(3C)-Tat-NLS-iFLP-6H	ara, 0.2%		8.64 / 123.4
16	164	pT7A3-MBP-(Tev)-GST-(3C)-Tat-NLS-iFLP-6H			8.64 / 123.4
17	162	pCold-MBP-(Tev)-GST-(3C)-Tat-NLS-iFLP-6H			8.64 / 123.4
18	211	pDBHisMBP-TaqMuts			5.40 / 134

Cloning

=======

3135  pCMV5-His-VH-IgG2b, His heavy

subclone from: 3135-3 pMiniT2

Vector Name: 3129 pCMV5-HA-VH-mIgG2b

Restriction Enzymes: BsrG1-Kpn1

Insert Length: 539

===

His Tag light

3001 pCMV5-His-VL-kappa

gene synthesis (not completed yet)

==========

3151 pCMV5-Myc-H-IgG2b, will not work

will not translate, need to fix sequence from BamH1-->EcoR1

subclone from 3003-H-1-PJET-1

Restriction Enzymes: BamH1-Kpn1

Vector Name: 2492 pCMV5-mIgG2b-HC

====

3075 pCMV5-Myc-L-kappa, completed

subclone from: 3003 pJet1.2-Myc 3003-L-5  light chain

Vector Name: 2493 pCMV5-mIgG-kLC

Restriction Enzymes: BamH1-Kpn1

Antibiotics: amp

====

for Myc tag Heavy, need to do 3076

3076 pCMV5-Myc-H-IgG2b (replaced by 3151)

PCR cloning from 

Vector Name: 3129 pCMV5-HA-VH-mIgG2b

PCR from 3003 pJet1.2-Myc heavy

3076-myc-BsrG1-F

atcatcTGTaCActccGAGGTGGACCTGGTTGAGTC

3076-myc-Nhe1-Kpn1-R

(CCACAGTGACCGTTTCTTCTGCTAGC GGTACC gatgat)

atcatcGGTACCGCTAGCAGAAGAAACGGTCACTGTGG

Restriction Enzymes: BsrG1-Kpn1: 

Insert Length: 403

=====

3009 pCMV5-Flag-H-IgG2, Flag heavy

subclone from: 3009-4-pMiniT2

Vector Name: 2492 pCMV5-mIgG2b-HC

Restriction Enzymes: BamH1-Kpn1

Insert Length: 539

Antibiotics: amp

====

3010 pCMV5-Flag-kappa, Flag light

need to fix from 3135-4-pMiniT2  (this 3135-4 clone is actually 3010) as-F_E12.ab1 template

it is actually 3010: missing 

5' end ATCATCGGATCCGCC

3' end: GGAGGAACAAAGCTCGAGATCAAGCGGGCTAGCGGTACCATCATC

using these oligos

3010 use 3135-4 as template
Positive control
3010-Flag-VL-3
3010-Flag-VL-10 

Full fragment:
-20 oC : 100 uM
5--> +45 ul H2O==> 10 uM
1 ul of 10 uM dilute to 20 ul H2O

3010-Flag-VL-1 F1 1 ul of 10 uM
3010-Flag-VL-2 R2 1 ul of 0.5 uM  
3010-Flag-VL-3 F3 1 ul of 0.5 uM
3010-Flag-VL-10   1 ul of 0.5 uM
3010-Flag-VL-11   1 ul of 0.5 uM
3010-Flag-VL-12  1 ul of 10 uM

Vector Name: 2493 pCMV5-mIgG-kLC

Restriction Enzymes: BamH1-Kpn1

=======

3129 pCMV5-HA-VH-mIgG2b, heavy

Vector Name: 2492 pCMV5-mIgG2b-HC

finished:

PCR from  3011 pJet1.2-HA Heavy chain

3129-BamH1-F

atcatcggatccAAGATCTAGAGCCGCCACCATG

3129-Kpn1-F

atcatcGTACCagacacggtaaccagtgttc

(gaacactggttaccgtgtctGGTACgatgat)

Clone into pMiniT2

Then subclone into 2492 pCMV5-mIgG2b-HC

Restriction Enzymes: BamH1 - Kpn1

==========

3130 pCMV5-HA-VL-mIgKappa HA light

Vector Name: 2493 pCMV5-mIgG-kLC

PCR from 3011 pJet1.2-HA light chain

clone: 3011-L-1 

3130-BamH1-R

atcatcggatccGCCGCCACCATGGACATGAGGGTC

3130-Kpn1-R

atcatcGGTACCccgcttcagttccagcttggttccgccgccaaatg

(catttggcggcggaaccaagctggaactgaagcggGGTACC gatgat)

Restriction Enzymes: BamH1-Kpn1

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3127 pET28b-qBeta-qBeta-6XHis

Primers

3127-Nde1-F
gatgat CATATGg GCCAAACTGGAAACCGTTAC
3127-HindIII-R
(gatcagctgaatccggcgtat AAGCTT atcatc)
gatgatAAGCTTatacgccggattcagctgatc

Insert size: 402

Vector: 3097 pET28b-qBeta
PCR using these primers and 3097 pET28b-qBeta as template,

digest with Nde1-HindIII, clone into Nde1-HindII digested  3097 pET28b-qBeta

ID	Name	DNA Size	Vector	Method	Status	Date	Enzyme	Expression	Note
940	pET28b-yPolyA-Pol	1713	pET28b	subclone from 2015 pEC2 using Nde1 -Xho1	completed
3105	pET28b-AaLS	699			finished	10/18/17	Nco1+ Xho1	has expression
3027	pCEH0-mIgG2b-HC
3097	pET28b-Qbeta	720			3097-2 sequence OK, subcloned into Nco1 + Sal1 into pET28b (Nco1+ Xho1)		pJet1.2: Nco1+ Xho1 Vector: Nco1+Sal1	no his tag
3127	pET28b-qBeta-qBeta-6XHis				PCR qbeta and clone back into 3097		Nde1-HindIII	C-His tag
3098	pET28b-AP205	592			good, subcloned into pET28b, no his tag, digestion with BamH1 to get c_his tag	transform Nico to test expression	Nco1+ Xho1
2490	pMiniT-mIgG2a-HC
2491	pCMV5-mIgG-kappa
3003	pJet1.2-Myc				submitted sequencing, light chain in pJet1.2 is good, finished need to do subcloning	10/18/17
812	pET30b-phoA
3082	pET28b-Cel1 947 pT7A30-6His-pMBP	837			pJet1.2, one clone sequencing verified, subcloned into pET28b failed, toxic gene	10/18/17	Nde1 + Xho1	toxic in  pET28b
3074	pET30b-HA-avitag-BirA				in pJet1.2 is good, subclone into pET28b, no his tag, need to subclone into pT7T30-Avi-BirA (still no his tag)
3011	pJet1.2-HA				H & L in pJet1.2 done, need to sublcone	10/10/2017
3010	pJet1.2-Flag				not working yet, need to do gene synthesis
3003	pCMV5-Myc-HC				pJet1.2 H & L sequenced				mAb 9E10 c-terminal c-myc tag
3001	pCMV5-His-VL-kappa				problem, can not get a good band from gene synthesis, should send it to IDT
3135	pCMV5-His-VH-IgG2b				pMiniT2 completed
938	PNGase F,	1145	pET30b	Nde1-Xho1	pJet1.2, sequencing is good, need to subclone into pET30b				C-His
939	Tte-Exo III	780	pET30b	Nde1-Xho1	pJet1.2 sequencing is good, need to subclone into pET30b				C-His
942	VVN endonuclease	716	pET28b-pMBP	Nco1-Xho1	pJet1.2 bad quality, need to restransform and make plasmid for sequencing				C-His
3122	Crm197, secreted	1743	pET30b	Nde1-Xho1	pJet1.2				C-His
931	PAK16D ligase	2267	pET28b	Nde1-HindIII-Xho1	pJet1.2 fragment 2 is good fragment 1  is also good				N-His
945	pT7A30-pMBP		pT7A3	Xba1-Nco1	PCR from 1281 pMAL-p5E or 1282 pMAL-p5E-AvPAL
947	pT7A30-6His-pMBP
948	pJet1.2-ccdb				sequenced, completed
944	pET30b-pfu-exo-
949	pET28b-GST-sfGFP-Myc-HA-Flag-V5		433 546
3075	pCMV5-Myc-L-kappa
3076	pCMV5-Myc-H-IgG2b
3010	pCMV5-Flag-kappa
3009	pCMV5-Flag-H-IgG2b
3129	pCMV5-HA-VH-mIgG2b
3130	2493 pCMV5-HA-VL-mIgKappa
3131	pCEL0-HA-VL-mIgKappa
3132	pCEH0-HA-VH-mIgG2b
	pCMV-PD1-AviTag-c6His
940	940 pET28b-yPolyA-Pol				sequenced, completed
824	pET28b-VVTopo
	3107 pFastbac-Cel1				GGATCC ACCATGAAGTTCTTGGTGAACGTGGCGCTGGTGTTCATGGTCGTGTACATCTCCTACATTT ACGCGTGGGGCAAAGAAGGCCACTACGCCGTGTGTAAGATTGCGGAGGGCTTTTTGTCGGA AGACGCATTGGGAGCGGTCAAAGCCTTGTTGCCGGACTACGCGGAAGGCGACTTGGCAGCC GTATGTAGCTGGGCCGACGAGATCAGACACAACTTTCACTGGAGATGGTCGGGCCCACTGC ATTACGTCGACACGCCGGATTACAGATGCAACTACGAGTACTGCCGCGACTGTCACGACTT CAGAGGCCACAAAGACATTTGCGTCACGGGCGCGATATACAACTACACGAAACAATTGACG TCGGGCTACCACAACAGTGGCTCCGAGATTCGATACAACCTCACGGAGGCCTTGATGTTCC TCTCGCATTTCATTGGCGACGTGCACCAACCGCTGCATGTGGGCTTTACGGGCGATGAAGG CGGAAATACGATCATTGTCCGTTGGTACCGCAGAAAGACCAACCTCCACCACATATGGGAC AACATGATCATCGACTCGGCGTTGAAGACCTACTACAACAGCGACCTGGCCATAATGATCC AGGCGATTCAAAGAAACATCACCGGCGATTGGTCCTTTGACATCAGCAGCTGGAAGAACTG TGCCAGTGACGACACTGCTTGTCCGAACCTATACGCGTCGGAGAGCATCTCGTTGGCCTGT AAATTTGCCTACAGAAATGCCACCCCCGGTTCGACGCTGGGCGACGACTACTTCTTGTCGC GATTGCCGATTGTTGAAAAACGCCTCGCCCAAGGCGGTATTAGATTGGCCGCCACCTTGAA CCGTATTTTTGCCTCGCAACCGAAAATCTCGC GAAACACGAAGACAAGAGAG CGAGAAG ACGACGCCGGTAGACTACATCGAGTGGTCGCCAT TGCAACAGT TCAGCGGAAGCCACCACC ATCACCACCATCATCACGGC ctcgag CAT CAT CAT CAT CAT CAC TGATAA     AAGCTT

Tag Antibodies

Name	Chain	Clone ID	Name	Vector	Status
vector	H	3129	pCMV5-HA-VH-mIgG2b	2492 pCMV5-mIgG2b-HC	3129-BamH1-F + 3129-Kpn1-F1
His	H+L, sc	3002	pJet1.2-Anti-His Tag 3D5	pJet1.2	3129-Kpn1-F1, sequenced, good, need to subclone into pET30b
His	L	3001	pCMV5-His-VL-kappa		could not clone, contract out to twist bio
	H	3135	3135-3-pMiniT2	pMiniT2	in pMiniT2 vector now, 3135-3-pMiniT2 sequenced, good,  need to subclone into 3129 pCMV5-HA-VH-mIgG2b using BsrG1-Kpn1
	L	3001	pCMV5-His-VL-kappa	3130 pCMV5-HA-VL-mIgKappa	gene synthesis from twist bio, to-do
	H	3135	pCMV5-His-VH-IgG2b	3129 pCMV5-HA-VH-mIgG2b	subclone into 3129 pCMV5-HA-VH-mIgG2b using BsrG1-Kpn1 from 3135-3-pMiniT2
Myc	L	3003	3003-L-5	pJet1.2	IL2 signal peptide, BamH1-Kpn1
	H	3003	3003-H-1	pJet1.2	need to use 3076-myc-BsrG1-F to fix frame shift
	L	3075	pCMV5-Myc-L-kappa	2493 pCMV5-mIgG-kLC	completed, subclone from 3003-L5 using BamH1-Kpn1
	H	3076	pCMV5-Myc-H-IgG2b	3129 pCMV5-HA-VH-mIgG2b	PCR from 3003-H-1 in pJet1.2, using 3076-myc-BsrG1-F  and  3076-myc-Nhe1-Kpn1-R
HA	L
	H
	L
	H
Flag	L
	H
	L
	H

Completed Clones

ID	Name

Need to Design

3031	GFP nanobody
	1 x TATA box binding protein TBP RP-54, order 5003
	2 x Schizosaccharomyces pombe Poly(U) Polymerase - Size: 100 Units ($170.00) EG-151, Order # 4971
	5 x Yeast Poly(A) Polymerase - Size: 100 Units ($290.00) EG-150, 4970 from 940 pET28b-yeastPolyA

Expression/Purification

ID	Name	Status	Antibiotics	Temp	pI/MW	Yield
	MMLV
	T7 RNA Polymerase
1950	pT7A3E-Fpg				8.47 / 31.9
927	pET28b-Cid1
3098	pET28b-AP205
	UDG
	Endonuclease VIII
3105	pET28b-AaLS
940	pET28b-yeastPolyA

NGS

Vendor	Cat	Product Name	Fragmentation	dA-Tailing	End Repair	Adaptor Ligation	Library Amplification	Size	Price
NEB			T7 Endonuclease I mutant Vibro vulnificus nuclease	Klenow Fragment (3´→ 5´ exo–) 1X NEBNext dA-Tailing Reaction Buffer: 10 mM Tris-HCl 10 mM MgCl2 50 mM NaCl 1 mM DTT 0.2 mM dATP pH 7.9 @ 25°C	End Repair Enzyme Mix: 10,000 units/ml T4 PNK 3,000 units/ml T4 DNA Polymerase 1X NEBNext End Repair Reaction Buffer: 50 mM Tris-HCl 10 mM MgCl2 10 mM DTT 1 mM ATP 0.4 mM dATP 0.4 mM dCTP 0.4 mM dGTP 0.4 mM dTTP pH 7.5 @ 25°C	T4 DNA Ligase USER Enzyme is a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII.
NuGen
Kapa

Purchasing & Shipping

Order #	Cat #	Name	Qty	Price	URL
4951	EG-1091	1 x HIV-I TAT (48 - 60) Protein Peptide , GRKKRRQRRRPPQ
4948	EG-151	2 x Schizosaccharomyces pombe Poly(U) Polymerase - Size: 100 Units ($170.00)

Designing

Clone ID	Name	URL	Note
934 EGF
933 pJet1.2-ccdb
931 AK16D ligase
929 pET28b-rtcBLigase

Cas9 proteins

1136 p8H-Exo-III Ecoli exonuclease III

1. Purification of 1032 pET6XHis-sso7d-wt-Taq sso7d-Taq

2. Clone 2474 pET30-HA-Tn5-6His

3, Clone 2443 pET28b-Tn5TPnase

4. QC Taq for qPCR 6. Transfection QC

5. Express 1881 pET30b-wt-Xho1 + 1878 pLysN-Plac-mXho1

6, Clone 

[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[1]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]] pET6XHis-sso7d-EP-Taq

[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[2]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]] pET-FastTaq-A743H

[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[3]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]] pET-FastTaq-E742A

2495 pMiniT-TP4-kLC-vr

2494 pMiniT-TP4-HC-vr

2493 pCMV5-mIgG2a-kLC

2492 pCMV5-mIgG2a-HC

2491 pMiniT-mIgG2a-kLC

2490 pMiniT-mIgG2a-HC

820 pHis-GST-HA-His
819 pHis-GST-myc-His
818 pHis-GST-His-Flag-His
821 pCold-GST-(3C)-T5Exo

Design thermostable luciferase,

Antibody Fusion  vector for IgG2a from 1162 pFUSE2-CLIg-hk 1161 pFUSE-CHIg-hG1 both of them from 1101

Protein A,

new bright GFP,

Dre

Cre-Flp

PD-1

PDL-1

Cas9

Cid1

T5-Exo

etc

Taq Polymerase primer-dimer QC

freeze dry new Taq, chemical modification, QC

design c-Myc 9E10 bacterial production plasmids

Purify important enzymes: for Sam

483 pET28b-TthPol (for RT-qPCR)

1995 Kfu

1811 VPol

1458 KOD

1483 T4 DNA Ligase

1464 T7 DNA Ligase

2409 pT7A3-Tat-dCre-(Tev)-Tat-NLS-iFLP-6H

1567 pT7A3-T5-Exo

1715 pT7A30-Pfu-sso7d pI is 8.47 / 98.7

1765 pT7A3-T4DNAPol   6.19 / 105.9

1722 pT7A3-sso7d-Native-ToughTaq(E708K)

1542 pT7A3-sso7d-Native-ToughTaq(E708K)

1497   pT7A3-ToughTaq(gene synthesis)

Purify important enzymes: for Paul

Transform competent cells with 1002 pLysN

DNA ladder

10X100 bp, 750 bp, 1500 bp, 2500 bp

4X250bp, 2X500 bp, 1 x 1000 bp

GR Green GR Red Stain to make dual color ladder

GR Green stain ladder from Ebay, GRRed stain of PCR product

ppt using PEG, reconsititute in 6X loading dye

make pMiniT vector

poly (U) polymerase 2252 pT7A3-Cid1,

gene synthesis shrimp alkaline phosphatase (SAP)

calf intestinal phosphatase (CIP)

bovine alkaline phosphatase (BAP)

Finished Project

927

pET28b-Cid1

978

sequencing is good, subcloned into pET28b: N-His tag

11/8/2017

Nde1+ Xho1

has expression