Paul.xu at 13:37, 23 September 2019

2019-09-23T13:37:55Z

New page

== Protein Expression Screening ==

{| border="1" cellspacing="0"
|-
| #
| Clone ID
| Name
| Induction
| His Tag
| pI MW
| Expression
|-
| 1
| 2596
| pT7A30-D7324-6H-CCMV
| <br/>
| <br/>
| 9.89 / 23.4
| <br/>
|-
| 2
| 2586
| pET28a-6H-D7324-AP205
| <br/>
| <br/>
| 9.88 / 17.6
| <br/>
|-
| 3
| 2595
| pDB.His.GST-D7324
| <br/>
| <br/>
| 6.90 / 31
| <br/>
|-
| 4
| 2594
| pDB.His.GST-SpyCatcher
| <br/>
| <br/>
| 5.75 / 39.3
| <br/>
|-
| 5
| 2593
| pDB.His.GST-SpyCatcher-AP205
| <br/>
| <br/>
| 5.99 / 53.6
| <br/>
|-
| 6
| 2585
| pT7A3-TRX-multi-Tags
| <br/>
| <br/>
| 4.67 / 49.7
| <br/>
|-
| 7
| 2587
| pT7A3-GST-multi-Tags
| <br/>
| <br/>
| 4.97 / 63.9
| <br/>
|-
| 8
| 2604
| pCold-MBP-multitag
| <br/>
| <br/>
| 4.88 / 80.6
| <br/>
|-
| 9
| 2580
| pET28a-Qbeta-Tat-6His
| <br/>
| <br/>
| 10.35 / 18.7
| <br/>
|-
| 10
| 2579
| pET28a-6H-Tat-AP205
| <br/>
| <br/>
| 10.54 / 18.3
| <br/>
|-
| 11
| 129
| pT7A30-TthMuts
| <br/>
| <br/>
| 5.87 / 91.8
| <br/>
|-
| 12
| 136
| pL6H-Tat-Cre
| <br/>
| <br/>
| 10.30 / 42.7
| <br/>
|-
| 13
| 135
| pQE-80L-HTNCre
| <br/>
| <br/>
| 10.30 / 42.8
| <br/>
|-
| 14
| 170
| pT7-MBP-(tev)-GST-(3c)-Tat-NLS-iFLP-6H
| <br/>
| <br/>
| 8.64 / 123.3
| <br/>
|-
| 15
| 169
| pBad-MBP-(Tev)-GST-(3C)-Tat-NLS-iFLP-6H
| ara, 0.2%
| <br/>
| 8.64 / 123.4
| <br/>
|-
| 16
| 164
| pT7A3-MBP-(Tev)-GST-(3C)-Tat-NLS-iFLP-6H
| <br/>
| <br/>
| 8.64 / 123.4
| <br/>
|-
| 17
| 162
| pCold-MBP-(Tev)-GST-(3C)-Tat-NLS-iFLP-6H
| <br/>
| <br/>
| 8.64 / 123.4
| <br/>
|-
| 18
| 211<br/>
| pDBHisMBP-TaqMuts<br/>
| <br/>
| <br/>
| 5.40 / 134<br/>
| <br/>
|}

== Cloning ==
<div>=======</div><div>3135  pCMV5-His-VH-IgG2b, His heavy</div><div>subclone from: 3135-3 pMiniT2</div><div>Vector Name: 3129 pCMV5-HA-VH-mIgG2b</div><div>Restriction Enzymes: BsrG1-Kpn1</div><div>Insert Length: 539</div><div>===</div><div>His Tag light</div><div>3001 pCMV5-His-VL-kappa</div><div><span style="color:#FF0000;">gene synthesis (not completed yet)</span><br/></div><div>==========</div><div>3151 pCMV5-Myc-H-IgG2b, <span style="color:#FF0000;">will not work</span></div><div>will not translate, need to fix sequence from BamH1-->EcoR1</div><div>subclone from 3003-H-1-PJET-1</div><div>Restriction Enzymes: BamH1-Kpn1</div><div>Vector Name: 2492 pCMV5-mIgG2b-HC</div><div>====</div><div>3075 pCMV5-Myc-L-kappa, completed</div><div>subclone from: 3003 pJet1.2-Myc 3003-L-5  light chain</div><div>Vector Name: 2493 pCMV5-mIgG-kLC</div><div>Restriction Enzymes: BamH1-Kpn1</div><div>Antibiotics: amp</div><div>====</div><div>for Myc tag Heavy, need to do 3076</div><div>3076 pCMV5-Myc-H-IgG2b (replaced by 3151)</div><div>PCR cloning from </div><div>Vector Name: 3129 pCMV5-HA-VH-mIgG2b</div><div>PCR from 3003 pJet1.2-Myc heavy</div><div>3076-myc-BsrG1-F</div><div>atcatcTGTaCActccGAGGTGGACCTGGTTGAGTC</div><div><br/></div><div>3076-myc-Nhe1-Kpn1-R</div><div>(CCACAGTGACCGTTTCTTCTGCTAGC GGTACC gatgat)</div><div>atcatcGGTACCGCTAGCAGAAGAAACGGTCACTGTGG</div><div>Restriction Enzymes: BsrG1-Kpn1: </div><div>Insert Length: 403</div><div>=====</div><div><br/></div><div>3009 pCMV5-Flag-H-IgG2, Flag heavy</div><div>subclone from: 3009-4-pMiniT2</div><div>Vector Name: 2492 pCMV5-mIgG2b-HC</div><div>Restriction Enzymes: BamH1-Kpn1</div><div>Insert Length: 539</div><div>Antibiotics: amp</div><div><br/></div><div>====</div><div>3010 pCMV5-Flag-kappa, Flag light</div><div>need to fix from 3135-4-pMiniT2  (this 3135-4 clone is actually 3010) as-F_E12.ab1 template</div><div>it is actually 3010: missing </div><div>5' end ATCATCGGATCCGCC</div><div>3' end: GGAGGAACAAAGCTCGAGATCAAGCGGGCTAGCGGTACCATCATC</div><div>using these oligos</div><div>
3010 use 3135-4 as template<br/>Positive control<br/>3010-Flag-VL-3<br/>3010-Flag-VL-10 

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

3010-Flag-VL-1 F1 1 ul of 10 uM<br/>3010-Flag-VL-2 R2 1 ul of 0.5 uM  <br/>3010-Flag-VL-3 F3 1 ul of 0.5 uM<br/>3010-Flag-VL-10   1 ul of 0.5 uM<br/>3010-Flag-VL-11   1 ul of 0.5 uM<br/>3010-Flag-VL-12  1 ul of 10 uM
</div><div><br/></div><div>Vector Name: 2493 pCMV5-mIgG-kLC</div><div>Restriction Enzymes: BamH1-Kpn1</div><div><br/></div><div>=======</div><div>3129 pCMV5-HA-VH-mIgG2b, heavy</div><div>Vector Name: 2492 pCMV5-mIgG2b-HC</div><div><br/></div><div>finished:</div><div>PCR from  3011 pJet1.2-HA Heavy chain</div><div><br/></div><div><br/></div><div>3129-BamH1-F</div><div>atcatcggatccAAGATCTAGAGCCGCCACCATG</div><div><br/></div><div>3129-Kpn1-F</div><div>atcatcGTACCagacacggtaaccagtgttc</div><div>(gaacactggttaccgtgtctGGTACgatgat)</div><div>Clone into pMiniT2</div><div><br/></div><div>Then subclone into 2492 pCMV5-mIgG2b-HC</div><div>Restriction Enzymes: BamH1 - Kpn1</div><div>==========</div><div>3130 pCMV5-HA-VL-mIgKappa HA light</div><div>Vector Name: 2493 pCMV5-mIgG-kLC</div><div>PCR from 3011 pJet1.2-HA light chain</div><div>clone: 3011-L-1 </div><div><br/></div><div>3130-BamH1-R</div><div>atcatcggatccGCCGCCACCATGGACATGAGGGTC</div><div><br/></div><div><br/></div><div>3130-Kpn1-R</div><div>atcatcGGTACCccgcttcagttccagcttggttccgccgccaaatg</div><div>(catttggcggcggaaccaagctggaactgaagcggGGTACC gatgat)</div><div><br/></div><div>Restriction Enzymes: BamH1-Kpn1</div><div>-------</div><div<br/></div><div>'''3127 pET28b-qBeta-qBeta-6XHis'''<br/></div><div>Primers</div><div>3127-Nde1-F<br/>gatgat CATATGg GCCAAACTGGAAACCGTTAC<br/>3127-HindIII-R<br/>(gatcagctgaatccggcgtat AAGCTT atcatc)<br/>gatgatAAGCTTatacgccggattcagctgatc<br/></div><div>Insert size: 402</div><div>Vector: 3097 pET28b-qBeta<br/>PCR using these primers and 3097 pET28b-qBeta as template,<br/></div><div>digest with Nde1-HindIII, clone into Nde1-HindII digested  3097 pET28b-qBeta<br/></div><div><br/></div><div><br/></div><div></div><div><br/></div>

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| <s>ID</s>
| <s>Name</s>
| <s>DNA Size</s>
| <s>Vector</s>
| <s>Method</s>
| <s>Status</s><br/>
| <s>Date</s>
| <s>Enzyme</s><br/>
| <s>Expression</s>
| <s>Note</s>
|-
| <s>940</s><br/>
| <s>pET28b-yPolyA-Pol</s><br/>
| <s>1713</s>
| <s>pET28b</s>
| <s> subclone from 2015 pEC2 using Nde1 -Xho1</s>
| <s>completed</s>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <s>3105</s><br/>
| <s> pET28b-AaLS</s><br/>
| <s>699</s>
| <br/>
|
|
<s>finished</s>

| 10/18/17
| <span style="font-family: Arial, Verdana, sans-serif; font-size: 12px; background-color: rgb(251, 251, 239);">Nco1+ Xho1</span>
| has expression
| <br/>
|-
| 3027<br/>
|  pCEH0-mIgG2b-HC<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <s>3097</s><br/>
| <s> pET28b-Qbeta</s><br/>
| <s>720</s>
| <br/>
| <br/>
| <s>3097-2 sequence OK, subcloned into Nco1 + Sal1 into pET28b (Nco1+ Xho1)</s><br/>
| <br/>
|
 pJet1.2: Nco1+ Xho1

Vector: Nco1+Sal1

| no his tag
| <br/>
|-
| 3127<br/>
| pET28b-qBeta-qBeta-6XHis<br/>
| <br/>
| <br/>
| <br/>
| PCR qbeta and clone back into 3097
| <br/>
| Nde1-HindIII
| C-His tag
| <br/>
|-
| <s>3098</s><br/>
| <s> pET28b-AP205</s><br/>
| <s>592</s>
| <br/>
| <br/>
|
<s>good, subcloned into pET28b, no his tag, digestion with BamH1 to get c_his tag</s>

| <s>transform Nico to test expression</s>
| <s>Nco1+ </s>Xho1<br/>
| <br/>
| <br/>
|-
| 2490<br/>
|  pMiniT-mIgG2a-HC<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 2491<br/>
|  pCMV5-mIgG-kappa<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <s>3003</s><br/>
| <s> pJet1.2-Myc</s><br/>
| <br/>
| <br/>
| <br/>
|
<s>submitted sequencing, light chain in pJet1.2 is good,</s>

<s>finished</s>

<s>need to do subcloning</s>

| 10/18/17
| <br/>
| <br/>
| <br/>
|-
| 812<br/>
|  pET30b-phoA<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <s>3082</s><br/>
|
<s> pET28b-Cel1</s>

<s>947 pT7A30-6His-pMBP</s>

| <s>837</s>
| <br/>
| <br/>
|
<s>pJet1.2, one clone sequencing verified,</s>

<s>subcloned into pET28b failed, toxic gene</s>

| <s>10/18/17</s>
| <s>Nde1 + X</s>ho1
| toxic in  pET28b
| <br/>
|-
| <span style="font-family: Arial, Verdana, sans-serif; font-size: 12px; white-space: nowrap;">3074</span><br/>
| <span style="font-family: Arial, Verdana, sans-serif; font-size: 12px; white-space: nowrap;">pET30b-HA-avitag-BirA</span><br/>
| <br/>
| <br/>
| <br/>
| in pJet1.2 is good, subclone into pET28b, no his tag, need to subclone into pT7T30-Avi-BirA (still no his tag)
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <s>3011</s>
| <s>pJet1.2-HA</s>
| <br/>
| <br/>
| <br/>
| <s>H & L in pJet1.2 done, need to sublcone</s>
| 10/10/2017
| <br/>
| <br/>
| <br/>
|-
| 3010<br/>
|  pJet1.2-Flag<br/>
| <br/>
| <br/>
| <br/>
| not working yet, need to do gene synthesis
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <s>3003</s>
| <s>pCMV5-Myc-HC</s><br/>
| <br/>
| <br/>
| <br/>
| <s>pJet1.2 H & L sequenced</s><br/>
| <br/>
| <br/>
| <br/>
| <span style="color:#FF0000;">mAb 9E10 c-terminal c-myc tag</span><br/>
|-
| <span style="color:#FF0000;">3001</span>
| <span style="color:#FF0000;">pCMV5-His-VL-kappa</span>
| <br/>
| <br/>
| <br/>
| <span style="color:#FF0000;">problem, can not get a good band from gene synthesis, should send it to IDT</span>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 3135 <br/>
| pCMV5-His-VH-IgG2b<br/>
| <br/>
| <br/>
| <br/>
| pMiniT2 completed
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 938
| PNGase F, 
| 1145
| pET30b<br/>
| Nde1-Xho1
| pJet1.2, sequencing is good, need to subclone into pET30b
| <br/>
| <br/>
| <br/>
| C-His<br/>
|-
| 939
| Tte-Exo III
| 780
| pET30b<br/>
| Nde1-Xho1<br/>
|
pJet1.2

sequencing is good, need to subclone into pET30b

| <br/>
| <br/>
| <br/>
| C-His
|-
| 942
| VVN endonuclease
| 716
| pET28b-pMBP<br/>
| Nco1-Xho1
|
pJet1.2

bad quality, need to restransform and make plasmid for sequencing

| <br/>
| <br/>
| <br/>
| C-His<br/>
|-
| 3122
| Crm197, secreted
| 1743
| pET30b<br/>
| Nde1-Xho1<br/>
| pJet1.2<br/>
| <br/>
| <br/>
| <br/>
| C-His
|-
| 931
| PAK16D ligase
| 2267
| pET28b
| Nde1-HindIII-Xho1<br/>
|
pJet1.2

fragment 2 is good

fragment 1  is also good

| <br/>
| <br/>
| <br/>
| N-His
|-
| 945<br/>
| pT7A30-pMBP<br/>
| <br/>
| pT7A3
| Xba1-Nco1<br/>
| PCR from 1281 pMAL-p5E or 1282 pMAL-p5E-AvPAL 
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 947<br/>
| pT7A30-6His-pMBP<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <s>948</s><br/>
| <s>pJet1.2-ccdb</s><br/>
| <br/>
| <br/>
| <br/>
| <s>sequenced, completed</s>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 944
| pET30b-pfu-exo-<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 949<br/>
| pET28b-GST-sfGFP-Myc-HA-Flag-V5<br/>
| <br/>
| 433 546<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 3075<br/>
| pCMV5-Myc-L-kappa<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 3076
| pCMV5-Myc-H-IgG2b<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 3010<br/>
| pCMV5-Flag-kappa<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 3009
| pCMV5-Flag-H-IgG2b<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 3129<br/>
|   pCMV5-HA-VH-mIgG2b<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 3130  <br/>
| 2493 pCMV5-HA-VL-mIgKappa<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 3131   <br/>
| pCEL0-HA-VL-mIgKappa<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 3132  <br/>
| pCEH0-HA-VH-mIgG2b<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| pCMV-PD1-AviTag-c6His<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <s>940</s>
| <s>940 pET28b-yPolyA-Pol</s><br/>
| <br/>
| <br/>
| <br/>
| <s>sequenced, completed</s>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 824<br/>
| pET28b-VVTopo<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <span style="color:#FF0000;">3107 pFastbac-Cel1</span><br/>
| <br/>
| <br/>
| <br/>
|
GGATCC<br/>ACCATGAAGTTCTTGGTGAACGTGGCGCTGGTGTTCATGGTCGTGTACATCTCCTACATTT ACGCGTGGGGCAAAGAAGGCCACTACGCCGTGTGTAAGATTGCGGAGGGCTTTTTGTCGGA AGACGCATTGGGAGCGGTCAAAGCCTTGTTGCCGGACTACGCGGAAGGCGACTTGGCAGCC GTATGTAGCTGGGCCGACGAGATCAGACACAACTTTCACTGGAGATGGTCGGGCCCACTGC ATTACGTCGACACGCCGGATTACAGATGCAACTACGAGTACTGCCGCGACTGTCACGACTT CAGAGGCCACAAAGACATTTGCGTCACGGGCGCGATATACAACTACACGAAACAATTGACG TCGGGCTACCACAACAGTGGCTCCGAGATTCGATACAACCTCACGGAGGCCTTGATGTTCC TCTCGCATTTCATTGGCGACGTGCACCAACCGCTGCATGTGGGCTTTACGGGCGATGAAGG CGGAAATACGATCATTGTCCGTTGGTACCGCAGAAAGACCAACCTCCACCACATATGGGAC AACATGATCATCGACTCGGCGTTGAAGACCTACTACAACAGCGACCTGGCCATAATGATCC AGGCGATTCAAAGAAACATCACCGGCGATTGGTCCTTTGACATCAGCAGCTGGAAGAACTG TGCCAGTGACGACACTGCTTGTCCGAACCTATACGCGTCGGAGAGCATCTCGTTGGCCTGT AAATTTGCCTACAGAAATGCCACCCCCGGTTCGACGCTGGGCGACGACTACTTCTTGTCGC GATTGCCGATTGTTGAAAAACGCCTCGCCCAAGGCGGTATTAGATTGGCCGCCACCTTGAA CCGTATTTTTGCCTCGCAACCGAAAATCTCGC GAAACACGAAGACAAGAGAG CGAGAAG ACGACGCCGGTAGACTACATCGAGTGGTCGCCAT TGCAACAGT TCAGCGGAAGCCACCACC ATCACCACCATCATCACGGC<br/>ctcgag CAT CAT CAT CAT CAT CAC<br/>TGATAA   

 AAGCTT

| <br/>
| <br/>
| <br/>
| <br/>
|}

== Tag Antibodies ==

{| border="1" cellspacing="1" cellpadding="1" style="width: 721px;"
|-
| style="width: 100px;" | Name
| style="width: 26px;" | Chain
| style="width: 47px;" | Clone ID
| style="width: 171px;" | Name
| style="width: 137px;" | Vector
| style="width: 345px;" | Status
|-
| style="width: 100px;" | vector
| style="width: 26px;" | H
| style="width: 47px;" | 3129 <br/>
| style="width: 171px;" | pCMV5-HA-VH-mIgG2b<br/>
| style="width: 137px;" | 2492 pCMV5-mIgG2b-HC<br/>
| style="width: 345px;" | 3129-BamH1-F + 3129-Kpn1-F1<br/>
|-
| style="width: 100px;" | His
| style="width: 26px;" | H+L, sc
| style="width: 47px;" | 3002<br/>
| style="width: 171px;" | pJet1.2-Anti-His Tag 3D5<br/>
| style="width: 137px;" | pJet1.2<br/>
| style="width: 345px;" | 3129-Kpn1-F1, sequenced, <span style="color:#FF0000;">good, </span>need to subclone into pET30b<br/>
|-
| style="width: 100px;" | His 
| style="width: 26px;" | L
| style="width: 47px;" | 3001 <br/>
| style="width: 171px;" | pCMV5-His-VL-kappa<br/>
| style="width: 137px;" | <br/>
| style="width: 345px;" | could not clone, contract out to twist bio
|-
| style="width: 100px;" | <br/>
| style="width: 26px;" | H<br/>
| style="width: 47px;" | 3135<br/>
| style="width: 171px;" | 3135-3-pMiniT2<br/>
| style="width: 137px;" | pMiniT2<br/>
| style="width: 345px;" |
in pMiniT2 vector now, 3135-3-pMiniT2 sequenced, <span style="color:#FF0000;">good,  </span>need to subclone into

3129 pCMV5-HA-VH-mIgG2b using BsrG1-Kpn1

|-
| style="width: 100px;" | <br/>
| style="width: 26px;" | L
| style="width: 47px;" | 3001 <br/>
| style="width: 171px;" | pCMV5-His-VL-kappa<br/>
| style="width: 137px;" | <span style="color: rgb(0, 0, 0); font-family: Arial, Verdana, sans-serif; font-size: 12px;">3130 pCMV5-HA-VL-mIgKappa</span><br/>
| style="width: 345px;" | gene synthesis from twist bio, <span style="color:#FF0000;">to-do</span>
|-
| style="width: 100px;" | <br/>
| style="width: 26px;" | H
| style="width: 47px;" | 3135
| style="width: 171px;" | <span style="background-color: rgb(251, 251, 239);">pCMV5-His-VH-IgG2b</span><br/>
| style="width: 137px;" | 3129 pCMV5-HA-VH-mIgG2b<br/>
| style="width: 345px;" | subclone into 3129 pCMV5-HA-VH-mIgG2b using BsrG1-Kpn1 from 3135-3-pMiniT2 <br/>
|-
| style="width: 100px;" | Myc<br/>
| style="width: 26px;" | L
| style="width: 47px;" | 3003 <br/>
| style="width: 171px;" | 3003-L-5<br/>
| style="width: 137px;" | pJet1.2<br/>
| style="width: 345px;" | IL2 signal peptide, BamH1-Kpn1<br/>
|-
| style="width: 100px;" | <br/>
| style="width: 26px;" | H
| style="width: 47px;" | 3003
| style="width: 171px;" | 3003-H-1<br/>
| style="width: 137px;" | pJet1.2<br/>
| style="width: 345px;" | need to use 3076-myc-BsrG1-F to fix frame shift
|-
| style="width: 100px;" | <br/>
| style="width: 26px;" | L
| style="width: 47px;" | 3075<br/>
| style="width: 171px;" | pCMV5-Myc-L-kappa<br/>
| style="width: 137px;" | 2493 pCMV5-mIgG-kLC<br/>
| style="width: 345px;" | completed, subclone from 3003-L5 using BamH1-Kpn1
|-
| style="width: 100px;" | <br/>
| style="width: 26px;" | H
| style="width: 47px;" | 3076<br/>
| style="width: 171px;" | pCMV5-Myc-H-IgG2b<br/>
| style="width: 137px;" | 3129 pCMV5-HA-VH-mIgG2b<br/>
| style="width: 345px;" | PCR from 3003-H-1 in pJet1.2, using 3076-myc-BsrG1-F  and  3076-myc-Nhe1-Kpn1-R<br/>
|-
| style="width: 100px;" | HA<br/>
| style="width: 26px;" | L
| style="width: 47px;" | <br/>
| style="width: 171px;" | <br/>
| style="width: 137px;" | <br/>
| style="width: 345px;" | <br/>
|-
| style="width: 100px;" | <br/>
| style="width: 26px;" | H
| style="width: 47px;" | <br/>
| style="width: 171px;" | <br/>
| style="width: 137px;" | <br/>
| style="width: 345px;" | <br/>
|-
| style="width: 100px;" | <br/>
| style="width: 26px;" | L
| style="width: 47px;" | <br/>
| style="width: 171px;" | <br/>
| style="width: 137px;" | <br/>
| style="width: 345px;" | <br/>
|-
| style="width: 100px;" | <br/>
| style="width: 26px;" | H
| style="width: 47px;" | <br/>
| style="width: 171px;" | <br/>
| style="width: 137px;" | <br/>
| style="width: 345px;" | <br/>
|-
| style="width: 100px;" | Flag<br/>
| style="width: 26px;" | L
| style="width: 47px;" | <br/>
| style="width: 171px;" | <br/>
| style="width: 137px;" | <br/>
| style="width: 345px;" | <br/>
|-
| style="width: 100px;" | <br/>
| style="width: 26px;" | H
| style="width: 47px;" | <br/>
| style="width: 171px;" | <br/>
| style="width: 137px;" | <br/>
| style="width: 345px;" | <br/>
|-
| style="width: 100px;" | <br/>
| style="width: 26px;" | L
| style="width: 47px;" | <br/>
| style="width: 171px;" | <br/>
| style="width: 137px;" | <br/>
| style="width: 345px;" | <br/>
|-
| style="width: 100px;" | <br/>
| style="width: 26px;" | H
| style="width: 47px;" | <br/>
| style="width: 171px;" | <br/>
| style="width: 137px;" | <br/>
| style="width: 345px;" | <br/>
|}

== ==

== Completed Clones ==

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| ID
| Name
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
|}

== Need to Design ==

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| <span style="background-color: rgb(251, 251, 239);">3031</span><br/>
| GFP nanobody
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| 1 x TATA box binding protein TBP RP-54, order 5003<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| 2 x Schizosaccharomyces pombe Poly(U) Polymerase - Size: 100 Units ($170.00) EG-151, Order # 4971<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| 5 x Yeast Poly(A) Polymerase - Size: 100 Units ($290.00) EG-150, 4970 from 940 pET28b-yeastPolyA<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|}

== Expression/Purification ==

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| ID
| Name
| Status
| Antibiotics
| Temp
| pI/MW
| Yield
| <br/>
|-
| <br/>
| MMLV<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| T7 RNA Polymerase
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 1950
| pT7A3E-Fpg<br/>
| <br/>
| <br/>
| <br/>
|  8.47 / 31.9<br/>
| <br/>
| <br/>
|-
| <span style="color:#FF0000;">927</span><br/>
| <span style="color:#FF0000;">pET28b-Cid1</span>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <span style="color:#FF0000;">3098</span><br/>
| <span style="color:#FF0000;">pET28b-AP205</span><br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| UDG
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Endonuclease VIII</span><br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <span style="color:#FF0000;">3105</span><br/>
| <span style="color:#FF0000;">pET28b-AaLS</span><br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <span style="color:#FF0000;">940 </span><br/>
| <span style="color:#FF0000;">pET28b-yeastPolyA</span><br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|}

== NGS ==

{| border="1" cellspacing="1" cellpadding="1" style="width:800px;"
|-
| Vendor
| Cat
| Product Name
| Fragmentation
| dA-Tailing<br/>
| End Repair<br/>
| Adaptor Ligation
| Library Amplification<br/>
| Size
| Price
|-
| NEB
| <br/>
| <br/>
|
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. 

| style="width: 80.2px;" | <br/>
| style="width: 80.2px;" | <br/>
| style="width: 1px;" | <br/>
|-
| NuGen
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| style="width: 80.2px;" | <br/>
| style="width: 80.2px;" | <br/>
| style="width: 1px;" | <br/>
|-
| Kapa
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| style="width: 80.2px;" | <br/>
| style="width: 80.2px;" | <br/>
| style="width: 1px;" | <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| style="width: 80.2px;" | <br/>
| style="width: 80.2px;" | <br/>
| style="width: 1px;" | <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| style="width: 80.2px;" | <br/>
| style="width: 80.2px;" | <br/>
| style="width: 1px;" | <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| style="width: 80.2px;" | <br/>
| style="width: 80.2px;" | <br/>
| style="width: 1px;" | <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| style="width: 80.2px;" | <br/>
| style="width: 80.2px;" | <br/>
| style="width: 1px;" | <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| style="width: 80.2px;" | <br/>
| style="width: 80.2px;" | <br/>
| style="width: 1px;" | <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| style="width: 80.2px;" | <br/>
| style="width: 80.2px;" | <br/>
| style="width: 1px;" | <br/>
|}

== Purchasing & Shipping ==

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Order #
| Cat #
| Name
| Qty
| Price
| URL
|-
| 4951<br/>
| EG-1091<br/>
| 1 x HIV-I TAT (48 - 60) Protein Peptide , GRKKRRQRRRPPQ <br/>
| <br/>
| <br/>
| <br/>
|-
| 4948<br/>
| EG-151<br/>
| 2 x Schizosaccharomyces pombe Poly(U) Polymerase - Size: 100 Units ($170.00) <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|}

== Designing ==

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Clone ID
| Name
| URL
| Note
|-
| 934 EGF<br/>
| <br/>
| <br/>
| <br/>
|-
| 933 pJet1.2-ccdb<br/>
| <br/>
| <br/>
| <br/>
|-
| 931 AK16D ligase<br/>
| <br/>
| <br/>
| <br/>
|-
| 929 pET28b-rtcBLigase<br/>
| <br/>
| <br/>
| <br/>
|}

Cas9 proteins

1136 p8H-Exo-III <span style="color: rgb(0, 0, 0); font-family: Arial, Verdana, sans-serif; font-size: 12px; white-space: nowrap;">Ecoli exonuclease III</span>

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 

[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[tel:808_1032|808|1032|||||||||||||||||||||||||||||||[1]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]] pET6XHis-sso7d-EP-Taq

[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[tel:810_1032|810|1032|||||||||||||||||||||||||||||||[2]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]] pET-FastTaq-A743H

[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[tel:809_1032|809|1032|||||||||||||||||||||||||||||||[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<br/>819 pHis-GST-myc-His<br/>818 pHis-GST-His-Flag-His<br/>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

<span style="color:#FF0000">'''Purify important enzymes: for Sam'''</span>

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)

<span style="color:#FF0000">Purify important enzymes: for Paul</span>

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 ==

{| border="1" cellspacing="1" cellpadding="1" style="width: 500px;"
|-
| 927<br/>
|  pET28b-Cid1<br/>
| 978<br/>
| <br/>
| <br/>
|
sequencing is good, subcloned into pET28b: N-His tag

| 11/8/2017
| Nde1+ Xho1
| has expression<br/><br/>
|}
[[Category:To Do]]

Tasks for This Week - Revision history

Paul.xu at 13:37, 23 September 2019