Paul.xu at 19:59, 17 July 2011

2011-07-17T19:59:15Z

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== '''Make  pAAV-MCS vector''' ==

'''Purpose:''' Make  pAAV-MCS vector 

'''Date: '''<strike>2011-6-21 to 2011-6-23</strike>

'''Notebook:''' 

'''Page:''' 

'''Clone ID:''' 254

'''Experimenters:''' Paul 

=== Annealing ===

Date: 

Anneal pAAV-MCS-T and PAAV-MCS-B at 950C for 2 minutes. Digest the vector pAAV-GFP (247) with EcoRI + XhoI 

Run gel and cut out vector,  purify using spin column

Ligate with annealed oligos for 2 hours 

Primer-F pAAV-MCS-T E7 aattc ACCGGT ggatcc tctaga gtcgac ctgcag GGTACC aagctt c Primer-R pAAV-MCS-B E8 tcgagaagcttGGTACCctgcaggtcgactctagaggatccACCGGTg 

and transform  STBL3

== '''Screening''' ==

 [[Image:252+254.JPG]] 

1: Marker 2: plenti-pka(ID, 300), #3,Nhe1+Xho1

3: plenti-pka(ID, 300), #4,Nhe1+Xho1

4: pAAV-MCS(ID, 254) ,#1, KpnI+Nco1

5: pAAV-MCS(ID, 254) ,#2, KpnI+Nco1

6: pAAV-MCS(ID, 254) ,#3, KpnI+Nco1

7: pAAV-MCS(ID, 254) ,#4, KpnI+Nco1

8: pAAV-MCS(ID, 254) ,#5, KpnI+Nco1

Sequencing: pLenti-pka(ID, 300), #3; pAAV-MCS(ID, 254) ,#2 

== '''Result intepretation/Summary''' ==
<pre>sequencing using CMV-F
NNNNNNNNNNNNNNNNNNNCNGANCTCGTTTAGTGNCCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGT
TTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCAT
TCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGT
AACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAA
GGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTTCTGGG
CAACGTGCTGGTCTGTGTGCTGCCCCNNNANTTTGNCTAAAAATTGGCATTCGAACTCCCATTGAANACCCNGTGNGTNC
NNTNNACCTGACCNCCTTTCTNNNNCNNNAAACAGGACGCTATGTCATAGCTGACGCTGTAGGTATATCANTTCNTTGTC
TNNTTTAGCTNTTAGCNANGNTNTTTCCCACNAANACCNCNNN

Score = 1040 bits (563), Expect = 0.0
Identities = 590/605 (98%), Gaps = 5/605 (1%)
Strand=Plus/Plus

Query 25 CTCGTTTAGTG-NCCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATA 83
||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 726 CTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATA 785

Query 84 GAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATT 143
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 786 GAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATT 845

Query 144 GGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCC 203
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 846 GGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCC 905

Query 204 ACAAAAAATGctttcttcttttaatatacttttttgtttatcttatttctaatactttcc 263
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 906 ACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCC 965

Query 264 ctaatctctttctttcAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCT 323
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 966 CTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCT 1025

Query 324 AAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATAT 383
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1026 AAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATAT 1085

Query 384 TTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATC 443
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1086 TTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATC 1145

Query 444 CAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAG 503
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1146 CAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAG 1205

Query 504 CTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTTCTGGGCAA 563
||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||
Sbjct 1206 CTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAA 1265

Query 564 CGTGCTGGTCTGTGTGCTGCCCCNNNANTTTGNCTAAA-AATTGGCATTCGAAC-TCCCA 621
||||||||||||||||||| ||| | |||| | ||| |||||| |||||||| || |
Sbjct 1266 CGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGC-AAAGAATTGGGATTCGAACATCG-A 1323

Query 622 TTGAA 626
|||||
Sbjct 1324 TTGAA 1328
</pre>
<pre>
Sequence using hGH-PA-R CCAGCTTGGTTCCCAATAGA

good
254_pAAV-MCS-hGH-PA-R_A12.ab1
NNNNNNNNNNNNNNNNNNNCNNNNNNNNTTGCNCNTTGCTCCNTACCACCCCCCTCCACCCCATAATATTATAGAAGGAC
ACCTAGTCAGACAAAATGATGCAACTTAATTTTATTAGGACAAGGCTGGTGGGCACTGGAGTGGCAACTTCCAGGGCCAG
GAGAGGCACTGGGGAGGGGTCACAGGGATGCCACCCGTAGATCTCTCGAGAAGCTTGGTACCCTGCAGGTCGACTCTAGA
GGATCCACCGGTGAATTCAATCGATGTTCGAATCCCAATTCTTTGCCAAAGTGATGGGCCAGCACACAGACCAGCACGTT
GCCCAGGAGCTGTGGGAGGAAGATAAGAGGTATGAACATGATTAGCAAAAGGGCCTAGCTTGGACTCAGAATAATCCAGC
CTTATCCCAACCATAAAATAAAAGCAGAATGGTAGCTGGATTGTAGCTGCTATTAGCAATATGAAACCTCTTACATCAGT
TACAATTTATATGCAGAAATATTTATATGCAGAAATATTGCTATTGCCTTAACCCAGAAATTATCACTGTTATTCTTTAG
AATGGTGCAAAGAGGCATGATACATTGTATCATTATTGCCCTGAAAGAAAGAGATTAGGGAAAGTATTAGAAATAAGATA
AACAAAAAAGTATATTAAAAGAAGAAAGCATTTTTTGTGGGCCTATAGACTCTATAGGCGGTACTTACGTCNNNCTTGGC
NCGGGGAAATCCGCGTTTCCNATGNNNCGTTCCCGNNNNNNNATTCNAA

rev-comp
TTNGAATNNNNNNNCGGGAACGNNNCATNGGAAACGCGGATTTCCCCGNGCCAAGNNNGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACATCGATT
EcoR1 AgeI BamH1 XbaI Sal1 Pst1 HindIII KpnI Xho1 BglII
GAATTC ACCGGT GGATCC TCTAGA GTCGAC CTGCAG GGTACC AAGCTT CTCGAG AGATCT

ACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTANGGAGCAANGNGCAANNNNNNNNGNNNNNNNNNNNNNNNNNNN

blast vs pAAV-MCS

Score = 1290 bits (698), Expect = 0.0
Identities = 714/727 (98%), Gaps = 2/727 (0%)
Strand=Plus/Plus

Query 15 CGGGAACGNNNCATNGGAAACGCGGATTTCCCCGNGCCAAGNNNGACGTAAGTACCGCCT 74
|||||||| ||| || |||||||| ||||||| |||||| ||||||||||||||||
Sbjct 12 CGGGAACGGTGCATTGG-AACGCGGA-TTCCCCGTGCCAAGAGTGACGTAAGTACCGCCT 69

Query 75 ATAGAGTCTATAGGCCCACAAAAAATGctttcttcttttaatatacttttttgtttatct 134
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 70 ATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCT 129

Query 135 tatttctaatactttccctaatctctttctttcAGGGCAATAATGATACAATGTATCATG 194
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 130 TATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATG 189

Query 195 CCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATA 254
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 190 CCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATA 249

Query 255 TTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGC 314
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 250 TTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGC 309

Query 315 TAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGG 374
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 310 TAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGG 369

Query 375 ATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTC 434
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 370 ATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTC 429

Query 435 CCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTG 494
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 430 CCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTG 489

Query 495 GGATTCGAACATCGATTGAATTCACCGGTGGATCCTCTAGAGTCGACCTGCAGGGTACCA 554
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 490 GGATTCGAACATCGATTGAATTCACCGGTGGATCCTCTAGAGTCGACCTGCAGGGTACCA 549

Query 555 AGCTTCTCGAGAGATCTACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGC 614
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Objct 550 AGCTTCTCGAGAGATCTACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGC 609

Query 615 CCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCAT 674
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 610 CCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCAT 669

Query 675 TTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTANGGAGCAA 734
|||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||
Sbjct 670 TTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAA 729

Query 735 NGNGCaa 741
| ||||
Sbjct 730 GGGGCAA 736

</pre>
 

 

== '''Conclusion''' ==

Clone pAAV-MCS(ID, 254) ,#2 is good 

=== ===

 =========== 

The  experiment described below by Ruili did not yield useful clone.

'''Make  pAAV-MCS vector'''

'''Purpose:''' Make  pAAV-MCS vector 

'''Date: '''<strike>2011-6-21 to 2011-6-23</strike>

'''Notebook:''' Ruili<--did not work 

'''Page:''' 002782

'''Clone ID:''' 254

'''Experimenters:''' <strike>Ruili </strike><--did not know how to make it to work. 

=== Annealing ===

Date: 2011-6-23

Anneal pAAV-MCS-T and PAAV-MCS-B at 950C for 2 minutes. Digest the vector pAAV-GFP (247) with EcoRI + XhoI 

Run gel and cut out vector combine and purify using spin column

Ligate with annealed DNA and transform Top10 <-- wrong, should be STBL3 

[[Image:PAAV-MCS-06-22-11-1.JPG]] 

<picture 1.> 

The picture is for the digested vector pAAV-GFP (247), the left is fast digestion buffer, the right is NEB buffer. 

[[Image:PAAV-MCS-06-23-11-2.JPG]] 

 

'''What are these? junk!'''

<picture 2.> 

1. the clone #1 after miniprep digest with KpnI + NCoI 

2. the clone # 2 after miniprep digest with KpnI + NCoI 

3. the clone # 3 after miniprep digest with KpnI + NCoI 

4. the clone #4 after miniprep digest with KpnI + NCoI

5. the clone # 5 after miniprep digest with KpnI + NCoI

6. the clone # 6 after miniprep digest with KpnI + NCol 

 

[[Image:PAAV-MCS-06-22-11-2.JPG]]

== ''''' ''''' ==

<picture 3> 

 

1. Marker; 

2. the digested vector pAAV-GFP (247)

Date: 2011-6-23

 

'''Junk'''

== '''PCR Screening''' ==

After subcloning, single out clones to do PCR screening to check if the insert is on the vector.

[[Image:PAAV-MCS-06-28-11-9.JPG]]

<picture 4> 

1. marker.

3. # 1 clone from PCR screening.

4. # 2 clone from PCR screening

5. # 3 clone from PCR screening

6. # 4 clone from PCR screening

7. # 5 clone from PCR screening

8. # 6 clone from PCR screening 

'''Junk''' 

--[[User:Ruili.su|Ruili.su]] 16:25, 29 June 2011 (EST)

After miniprep, then digest with i) KpnI + NCoI or ii) KPnI + NdeI to check if  the inser is on the cloned vector. 

[[Image:PAAV-MCS-06-30-11-4.JPG]] 

<picture 5>

1. marker. 

3. # 4 clone, after miniprep, digest with KPnI + NdeI 

4. # 5 clone, after miniprep, digest with KPnI + NdeI

5. # 6 clone, after miniprep, digest with KPnI + NdeI

 

 

== '''Result intepretation/Summary''' ==

*<strike>Picture 1. shows restriction digestion for the vector pAAV-GFP (247) are all good with fast digestion buffer and NEB buffer. </strike>
*<strike>The clone 1--- 6 (picture 2) have no inserts released after diestion with KpnI + NCoI. </strike>
*<strike>Picture 3: the clone #1 through # 6 (line 3 ---8) indicate that MCS was inserted to pAAV.</strike>
*<strike>Picture 5: the clone # 5 and # 6 (line 4 and 5) show the MCS is on the vector.</strike>

'''Junk'''

== '''Conclusion''' ==

*<strike> Picture 2 shows that the clones are self ligation and have no insert. </strike>
*<strike> Picture 3 shows the sub-cloning is successful.</strike>
*<strike>Picture 5 shows we can furtherly do miniprep to store the cloned vector.</strike>

<strike>Junk</strike> --[[User:Ruili.su|Ruili.su]] 16:32, 30 June 2011 (EST)

=== ===

 --[[User:Ruili.su|Ruili.su]] 17:21, 24 June 2011 (EST)

The above description is junk, none is working.

--[[User:Paul.xu|Paul.xu]] 12:24, 17 July 2011 (EST)

[[Category:Cloning]]

PAAV-Mcs - Revision history

Paul.xu at 19:59, 17 July 2011