PAAV-Mcs

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Make  pAAV-MCS vector

[edit]

Purpose: Make  pAAV-MCS vector 

Date: 2011-6-21 to 2011-6-23

Notebook:

Page:

Clone ID: 254

Experimenters: Paul

Annealing

[edit]

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

[edit]



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

[edit]
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

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



Conclusion

[edit]

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

[edit]


===========

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

Make  pAAV-MCS vector

Purpose: Make  pAAV-MCS vector 

Date: 2011-6-21 to 2011-6-23

Notebook: Ruili<--did not work

Page: 002782

Clone ID: 254

Experimenters: Ruili <--did not know how to make it to work.

Annealing

[edit]

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


<picture 1.>

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



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



[edit]

<picture 3>


1. Marker;

2. the digested vector pAAV-GFP (247)

Date: 2011-6-23


Junk

PCR Screening

[edit]

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

<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

--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.


<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

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

Junk

Conclusion

[edit]
  •  Picture 2 shows that the clones are self ligation and have no insert.
  •  Picture 3 shows the sub-cloning is successful.
  • Picture 5 shows we can furtherly do miniprep to store the cloned vector.

Junk
--Ruili.su 16:32, 30 June 2011 (EST)

[edit]


--Ruili.su 17:21, 24 June 2011 (EST)

The above description is junk, none is working.

--Paul.xu 12:24, 17 July 2011 (EST)

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