Ligation plans statues 8.3:

DKK1: Plan is to lift the DKK1 sequence, with newly added 3'-KPNI and 5'-HINDIII cutting site on flanks.

Primers: 1. DKK1 5 HindIII- GACCGCAAGCTTTTGCAGGATCCCATCGATTC 2. DKK1 3 KpnI- GTGGTACCATGGACTCACTATAGTTCTAGAGG

So the PCR product will look like:

GACCGCA^AGCTT-DKK1-GTGGTAC^C

DKK1 flanking Sequences (between ECORI and XhoI):

TTGCAGGATCCCATCGATTCGAATTCAAGGCCTCTCGAGCCTCTAGAACTATAGTGAGT

Area of insertion to H2B-VFP: A^agcttggtac^c

PCR images:

Post cutting images (insert #4+#5, Vector #6 + #7):

Possible problems:

a. The insert is not being cut (too large amount of DNA?). b. The vector is not being cut (Too large amount of DNA?). c. The sequence of the insert is wrong (Need to sequence with internal primer).

What to do? 1. Order DKK1 primer to check out insert. 2. Try cutting using <0.2ug of PCR(insert) DNA and <1ug plasmid (vector) DNA for reaction.

GAL's VFP halves: Plan is to lift two halves of VFP, SEPRATELY, into the FUW-MBP clone (GP1), immediately after the MBP sequence. Also, lift the full YFP from FUW-YFP into GP1.

Primers:

For first half of VFP, and full YFP: 1. MLU-YFP1: tataAcgcgtGTGAGCAAGGGCGAGGAG 2. YFP1-xba: TATAtctagaCATGATATAGACGTTGTGGCTGTTG

For second half of VFP: 3. Mlu-YFPend: tataAcgcgtGCCGACAAGCAGAAGAACG 4. YFPend-xba: TATAtctAGActtgtacaGCTCGTCCATGC

Target: GP1 (MBP inserted after FUW in FUW-YFP).

Is the product of lifting MBP from PAT224 using the following primers:

128: 5' EcoR1-MBP: tatagaattcccaccATGGGGAAAACTGAAGAAGGTAAAC 129: 3' MBP-Bam-G4S-Mlu-xba1-mfe: TATAcaattgTTAtctagaagaACGCGTagatcctcctcctcCggatccAGTCTGCGCGTCTTTCAG

Mfe- CAATTG xbaI- TCTAGA Mlu- ACGCGT G4S- BamHI- GGATCC

Templates:

YFP1 (AA1-154): ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATG

YFPend(154-end): GCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCtgtacaag

YFP (From Fuw-YFP)- So actually this template can be used for all three reactions.

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG

-- InbarC - 07 Mar 2011