Paul.xu at 22:16, 13 February 2018

2018-02-13T22:16:56Z

New page

== Cloning ==

{| border="1" cellspacing="1" cellpadding="1" style="width: 675px;"
|-
| ID
| #
| Name
| style="width: 132px;" | Status
| style="width: 193px;" | Note
| style="width: 111px;" | Note2
| style="width: 144px;" | Note3
|-
| 3129
| 1
| pMiniT2 
| style="width: 132px;" | sequencing
| style="width: 193px;" | sequncing: good, but looks like missing ut digestion does show Kpn1 -BamH1 sites 
| style="width: 111px;" | target construct pCMV5-HA-VH-mIgG2b 
| style="width: 144px;" | need to subclone 2492 pCMV5-mIgG2b-HC
|-
| 3075
| 1
| pCMV
| style="width: 132px;" | sequencing
| style="width: 193px;" | good
| style="width: 111px;" | good
| style="width: 144px;" | 
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Date:2/2/2018

Digest of PCR products

'''3010 pCMV5-Flag-kappa, Flag light'''

Gene Synthesize

need to fix from 3135-4-pMiniT2 >as-F_E12.ab1 template it is actually 3010: missing 5' end ATCATCGGATCCGCC 3' end: GGAGGAACAAAGCTCGAGATCAAGCGGGCTAGCGGTACCATCATC using these oligos

Insert: 1 ul of 10 uM 3010-Flag-VL-1C4ATCATCGGATCCGCCGCCACCATG 1 ul of 1 uM 3010-Flag-VL-2C5AGAGCAGCAGGAGCCCCAGGAGCTGAGCGGGGACCCTCATGTCCATGGTGGCGGCGGA 1 ul of 1 uM 3010-Flag-VL-11D2CGCTCCTTACACCTTTGGCGGAGGAACAAAGCTCGAGATCAAGCGGGCTAGCGGTACC 1 ul of 10 uM 3010-Flag-VL-12D3GATGATGGTACCGCTAGCCCGC

'''3135-4-pMiniT2 as template'''

Vector Name: 2493 pCMV5-mIgG-kLC

Restriction Enzymes: BamH1-Kpn1

[[File:3010 3003 3011 H&L.JPG|RTENOTITLE]]

[[File:3151 pMiniT2 TEST.JPG|RTENOTITLE]]

[[File:Gel purfication digest.JPG|RTENOTITLE]]

[[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]]) 17:16, 13 February 2018 (EST)== 17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST) [[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]]) 17:16, 13 February 2018 (EST)==

Date:2/5/2018 subcloning subclone of 3009-4_2492

[[File:3009-4 2492.JPG|RTENOTITLE]]

Date:2/5/2018

PCR cloning Digest of Vector 2492_2493

[[File:2492 2493.JPG|RTENOTITLE]]

Date:2/5/2018

BamHI Test

[[File:BamhI test.JPG|RTENOTITLE]]

Date:2/5/2018 PCR Cloning of 3129_3130 3130 Fix and 3021 test

'''3129 pCMV5-HA-VH-mIgG2b, heavy'''

Vector Name: 2492 pCMV5-mIgG2b-HC

PCR from '''3011 pJet1.2-HA''' Heavy chain

Clone into pMiniT2

Subclone into 2492 pCMV5-mIgG2b-HC

Restriction Enzymes: BamH1 - Kpn1

10:30, 6 February 2018 (EST)10:30, 6 February 2018 (EST)

'''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

Restriction Enzymes: BamH1-Kpn1

10:30, 6 February 2018 (EST)

3130-Fix and 3021 are for sequencing test,because the result showed no primering

Annealing:55oC

Note:

3129-BamH1-F atcatcggatccAAGATCTAGAGCCGCCACCATG

3129-Kpn1-F atcatcGTACCagacacggtaaccagtgttc (gaacactggttaccgtgtctGGTACgatgat)

3130-BamH1-R atcatcggatccGCCGCCACCATGGACATGAGGGTC

3130-Kpn1-R atcatcGGTACCccgcttcagttccagcttggttccgccgccaaatg (catttggcggcggaaccaagctggaactgaagcggGGTACC gatgat) [[User:Crm|Crm]] ([[User_talk:Crm|talk]]) 10:30, 6 February 2018 (EST)

'''Figure description'''

3129 # 1,2,: Use Platinum HiFi PCR 2X mix

3130 # 3: Use DreamTaq 2X Mix

3130-fix: Dream Taq 2X mix, using pMiniT2 F & R primers, testing why we got no priming in sequencing exp

3021 using pMiniT2 F & R primers, testing why we got no priming in sequencing exp

[[File:3129 3130.JPG|RTENOTITLE]]

[[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]]) 17:16, 13 February 2018 (EST)== 17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)[[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]])== [[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]]) 17:16, 13 February 2018 (EST)==

Date:2/6/2018

PCR Cloning of '''3129'''

'''Figure despriction''' Template: 3011 H in pJet1.2, sequenced, annealing: 55 oC 30'', Extension 72 oC 30''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane
| Primers
| Enzyme
|-
| Ladder
| 
|
|-
| 1
| 3129-F + 3129-R 
| DreamTaq 
|-
| 2
| pJet1.2 F + R
| DreamTaq
|-
| 3
| 3129-F + 3129-R 
| Plantimum PCR Supermix HF 
|-
| 4
| pJet1.2 F + R 
| Plantimum PCR Supermix HF 
|-
| 5
| 3129-F + 3129-R 
| Q5 DNA polymerase
|-
| 6
| pJet1.2 F + R 
| Q5 DNA polymerase 
|}

[[File:PCR for 3011(Paul).JPG|RTENOTITLE]]

[[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]]) 17:16, 13 February 2018 (EST)== 17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)~=== [[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]]) 17:16, 13 February 2018 (EST)==

Date;2/6/2018

PCR: Test the result of sequencing; annealing: 55 oC 30'', Extension 72 oC 30''

'''Figure Despriction'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| #
| template
| 2X PCR Mix
| Primer
|-
| 1
| + Control---3011 H
| DreamTaq
| 3129 F&R
|-
| 2
| 3021-2
| <div>DreamTaq</div>
| pMiniT2
|-
| 3
| 3021-1
| DreamTaq
| pMiniT2
|-
| 4
| 3130-Fix-1
| DreamTaq
| pMiniT2
|}

'''Result: the 3021-1,3021-2,3130-Fix-1 are contaminted, so we abandoned them.'''

[[File:Test for no primering sequencing.JPG|RTENOTITLE]]

Date:2/6/2018

'''Figure Despriction'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| #
| Template
| 2X PCR Mix
| Primer
|-
| 1
| 3011-H (control)
| DreamTaq
| 3129 F&R
|-
| 2
| 3011 L
| Plantinum PCR Super mix HiFi
| 3130 F&R
|-
| 3
| 3011 L
| Q5 DNA Polymerase
| 3130 F&R
|-
| 4
| 3011 H
| Plantinum PCR Super mix HiFi 
| 3129 F&R
|-
| 5
| 3011 H
| Plantinum PCR Super mix HiFi 
| 3129 F&R
|}

[[File:PCR of 3011L 3011H.JPG|RTENOTITLE]] 

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[[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]]) 17:16, 13 February 2018 (EST)== 17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)[[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]]) 17:16, 13 February 2018 (EST)=== [[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]]) 17:16, 13 February 2018 (EST)==

Date:2/7/2018

'''Figure Description'''

PCR of different Taq enzymes

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| #
| Template
| 2X PCR Mix
| Primer
|-
| 1
| 3011 H(+)
| DreamTaq
| pMini T2
|-
| 2
| 3011 H
| DreamTaq
| pMIniT2
|-
| 3
| 3011 H
| PCR MIx
| pMIniT2
|-
| 4
| 3011 H
| 0.1Taq
| pMIniT2
|-
| 5
| 3011 H
| DreamTaq
| 3129 F&R
|}

'''Results: We didn't get good results.'''

[[File:Test of different products.JPG|RTENOTITLE]]

'''Figure Despriction'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| #
| Template
| 2X PCR
| Primer
|-
| 1
| 3011 H
| DreamTaq
| 3129 F&R
|-
| 2
| 3011 H
| DreamTaq
| pMiniT2
|-
| 3
| 3011 H
| PCR Mix
| pMiniT2
|-
| 4
| 3011 L
| 0.1 Taq
| pMiniT2
|-
| 5
| 3011 L
| PCR Mix
| 3130 F&R
|-
| 6
| 3011 L
| 0.1 Taq
| 3130 F&R
|}

[[File:PCR of different enzymes 2.JPG|RTENOTITLE]]

'''Figure Despriction: '''

'''Annealing:55oC, Extension:30s,'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| #
| Template
| 2X PCR Mix
| Primers
|-
| 1
| 3011 L
| phusion
| pMini T2 F&R
|-
| 2
| <div>3011 L</div>
| phusion
| pMini T2 F&R 
|-
| 3
| 3011 L 
| phusion
| pMini T2 F&R 
|-
| 4
| 3011 L 
| phusion
| pMini T2 F&R 
|-
| 5
| 3011 L
| kfu
| pMini T2 F&R 
|-
| 6
| 3011 L
| kfu
| pMini T2 F&R 
|-
| 7
| 3011 L 
| kfu
| pMini T2 F&R 
|-
| 8
| 3011 L 
| kfu
| pMini T2 F&R 
|-
| 9
| 3011 L 
| KOD
| pMini T2 F&R 
|-
| 10
| 3011 L 
| KOD
| pMini T2 F&R 
|-
| 11
| 3011 L 
| KOD
| pMini T2 F&R 
|-
| 12
| 3011 L
| KOD
| pMini T2 F&R 
|-
| 13
| 3011 L
| 0.5ul Taq+ 0.5ul pfu
| pMini T2 F&R 
|-
| 14
| 3011 L
| 0.5ul Taq+ 0.5ul pfu 
| pMini T2 F&R 
|-
| 15
| 3011 L(-)
| no DNA polymerase
| pMini T2 F&R 
|}

[[File:Polymerase QC for using PCR.JPG|RTENOTITLE]]

'''Results: The enzymes may not work.'''

[[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]]) 17:16, 13 February 2018 (EST)== 17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)[[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]]) 17:16, 13 February 2018 (EST) [[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]]) 17:16, 13 February 2018 (EST)==

'''Date:2/8/2018''' '''Figure Despriction'''

'''Digestion of 3011 L_3011 H'''

http://www.excellgen.net/eln/images/3/3e/Digestion_of_3011_L_3011_H.JPG

'''Result:#1,#2,#4,#5,#7,#8 has bands. So we did PCR for these samples.'''

'''Figure Despriction'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| #
| Tempate
| 2X PCR Mix
| Primer
|-
| 1
| 3011 L (For 3130)
| 0.1 Taq
| pMiniT2 F&R
|-
| 2
| 3011 L
| 0.1 Taq 
| pMiniT2 F&R 
|-
| 4
| 3011 L
| 0.1 Taq 
| pMiniT2 F&R 
|-
| 5
| 3011 L
| 0.1 Taq 
| pMiniT2 F&R 
|-
| 6
| 3011 H(For 3129)
| 0.1 Taq 
| pMiniT2 F&R 
|-
| 7
| 3011 H
| 0.1 Taq 
| pMiniT2 F&R 
|-
| 8
| 3011 H
| 0.1 Taq 
| pMiniT2 F&R 
|-
| 9
| 3011 H
| 0.1 Taq 
| pMiniT2 F&R 
|-
| 10
| 3011 H(+)
| 0.1 Ta1
|}

[[File:PCR of Mini Prep for 3130 and 3129.JPG|RTENOTITLE]]

Result: we picked #1,#8 to do sequencing. #1 is no primering.#8 is missing C in Kpn I.

Friday,2/9/2018, we ordered another sequencing for #4.

'''Figure Despriction'''

'''Subclone of 3009'''

Subclone from 3009-4-pMiniT2, 

Vector from 2492

[[File:Subclone 3009-4 2492.JPG|RTENOTITLE]]

Result:cut the bands and do ligation.

[[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]]) 17:16, 13 February 2018 (EST)== 17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)17:16, 13 February 2018 (EST)=== [[User:Paul.xu|Paul.xu]] ([[User talk:Paul.xu|talk]]) 17:16, 13 February 2018 (EST)==

Date:2/9/2018

'''Protein purfication: 15 samples.'''

'''Use buffer B to elute'''

'''Concentration:'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| #
| Concentration
|-
| 1
| -0.53
|-
| 2
| 0.48
|-
| 3
| 1.59
|-
| 4
| 2.24
|-
| 5
| 1.26
|-
| 6
| 1.87
|-
| 7
| 1.17
|-
| 8
| 0.63
|-
| 9
| 0.50
|-
| 10
| 0.30
|-
| 11
| 0.53
|-
| 12
| 0.29
|-
| 13
| 0.14
|-
| 14
| 0.11
|-
| 15
| 0.09
|}

'''PCR Gene synthesis -----3010'''

{| border="1" cellspacing="1" cellpadding="1" style="width: 778px;"
|-
| #
| Tempate
| style="width: 111px;" | Oligo
| style="width: 174px;" | 2X PCR Mix
| style="width: 46px;" | Primer
| style="width: 307px;" | Note
|-
| 1
| 3135-4
| style="width: 111px;" | 3010-D1,D2,D3 
| style="width: 174px;" | Platinum 2X SuperMix HiFi
| style="width: 46px;" | C4,C5,C6
| style="width: 307px;" | C4--10uM,C5,C6,D1,D3--0.5uM,D3--10uM
|-
| 2
| 3135-4  
| style="width: 111px;" | 3010-D1,D2,D3 
| style="width: 174px;" | Platinum 2X SuperMix HiFi 
| style="width: 46px;" | C4,C5,C6 
| style="width: 307px;" | C4--10uM,C5,C6,D1,D3--0.5uM,D3--10uM
|-
| 3
| 3135-4  
| style="width: 111px;" | 3010-D1,D2,D3 
| style="width: 174px;" | Platinum 2X SuperMix HiFi 
| style="width: 46px;" | C4,C5,C6 
| style="width: 307px;" | C4--10uM,C5,C6,D1,D3--0.5uM,D3--10uM 
|-
| 4
| 3135-4 (+)
| style="width: 111px;" | 3010-C6,D1 
| style="width: 174px;" | Platinum 2X SuperMix HiFi 
| style="width: 46px;" | C6.D1
| style="width: 307px;" | C6,D1---10uM
|}

[[File:PCR of 3010 gene synthesis.JPG|RTENOTITLE]]

'''PCR Gene synthesis -----3001'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| #
| Oligo
| 2X PCR Mix
| Primer
| Note
|-
| 1
| 3001 Oligo Mix
| Q5
| E6,F5
| 
|-
| 2
| 3001 Oligo Mix 
| Q5
| E6,F5
| 
|-
| 3
| 3001 Oligo Mix 
| Q5
| E6,F5
| 
|-
| 4
| 3001 Oligo Mix 
| Q5
| E6,F5
| 
|}
<div></div
[[File:PCR of 3001 gene synthesis.JPG|RTENOTITLE]]

'''Result: we didn't synthesiz ''''''3001'''''' successfully.'''

'''Colony screening for 3009 subclone'''

'''Figure Desperction'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| #
| Template
| 2X PCR Mix
| Primer
|-
| 1
| 3009-1
| 0.1Taq
| pMiniT2 F&R
|-
| 2
| 3009-2
| .....
| ............
|-
| 3
| 3009-3
| .....
| .............
|-
| 4
| 3009-4
| .....
| ..............
|-
| 5
| 3009-5
| .....
| ..............
|-
| 6
| 3009-6
| .....
| .............
|-
| 7
| 3009-7
| .....
| .............
|-
| 8
| 3009-8
| .....
| ..............
|-
| 9
| 3009-9
| .....
| .................
|-
| 10
| 3009-10
| .....
| ....................
|-
| +
| 3011 L
| 0.1Taq
| pMiniT2 F&R
|}

[[File:Colony screening.JPG|RTENOTITLE]]
</div>[[Category:My ELN]]

Yijia LI 2 2 2018 - Revision history

Paul.xu at 22:16, 13 February 2018